Identification of metabolites of liquiritin in rats by UHPLC-Q-TOF-MS/MS: Metabolic profiling and pathway comparison: In vitro and in vivo

Article abstract of DOI:10.1039/c7ra13760e

Liquiritin (LQ), the main bioactive constituent of licorice, is a common flavoring and sweetening agent in food products and has a wide range of pharmacological properties, including antidepressant-like, neuroprotective, anti-cancer and anti-inflammatory properties. This study investigated the metabolic pathways of LQ in vitro (rat liver microsomes) and in vivo (rat model) using ultra high-performance liquid chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS/MS). Moreover, supplementary tools such as key product ions (KPIs) were employed to search for and identify compounds. As a result, 56 in vivo metabolites and 15 in vitro metabolites were structurally characterized. Oxidation, reduction, hydrolysis, methylation, acetylation, and sulfate and glucuronide conjugation were determined to be the major metabolic pathways of LQ, and there were differences in LQ metabolism in vitro and in vivo. In addition, the in vitro and in vivo metabolic pathways were compared in this study.

Full text of DOI:10.1039/c7ra13760e

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Identification of metabolites of liquiritin in rats by
UHPLC-Q-TOF-MS/MS: metabolic profiling and
pathway comparison in vitro and in vivo†
Cite this: RSC Adv., 2018, 8, 11813
*
Xia Zhang, Caijuan Liang, Jintuo Yin, Yupeng Sun and Lantong Zhang
Liquiritin (LQ), the main bioactive constituent of licorice, is a common flavoring and sweetening agent in
food products and has a wide range of pharmacological properties, including antidepressant-like,
neuroprotective, anti-cancer and anti-inflammatory properties. This study investigated the metabolic
pathways of LQ in vitro (rat liver microsomes) and in vivo (rat model) using ultra high-performance liquid
chromatography coupled with hybrid triple quadrupole time-of-flight mass spectrometry (UHPLC-Q-
TOF-MS/MS). Moreover, supplementary tools such as key product ions (KPIs) were employed to search
for and identify compounds. As a result, 56 in vivo metabolites and 15 in vitro metabolites were
structurally characterized. Oxidation, reduction, hydrolysis, methylation, acetylation, and sulfate and
glucuronide conjugation were determined to be the major metabolic pathways of LQ, and there were
differences in LQ metabolism in vitro and in vivo. In addition, the in vitro and in vivo metabolic pathways
were compared in this study.
Received 30th December 2017
Accepted 3rd March 2018
DOI: 10.1039/c7ra13760e
rsc.li/rsc-advances
seeds, soy sauce, etc., are being extensively researched.9,10
Among the biologically active compounds in licorice, liquiritin
1. Introduction
(LQ, Fig. 1) is the dominant component and is considered to be
the major active ingredient.11
Licorice root or Radix glycyrrhizae, a Chinese materia medica
(CMM), is widely used to invigorate the spleen, replenish the Qi,
dispel heat and remove toxic substances.1 In modern medical
terms, the biologically active compounds in licorice are tri-
terpene, saponins, avonoids, polysaccharides and phenolic
compounds,2 making licorice root a source of medicine and
food. These compounds exhibit several important pharmaco-
logical activities, including anti-viral,3 anti-oxidant,4 anti-
bacterial,5 anti-inammatory6 and anti-HIV7 activities. More-
over, licorice and its extract are widely used in health foods
because of the physiological activities of these substances.8
Currently, the use of licorice and its extract in grain products,
oil products, meat products, beverages, candy, jelly, dried fruit,
Drug metabolism may lead to detoxication and/or activa-
tion reactions, and studies of drug metabolism can aid the
identication of active compounds and explain the mecha-
nisms of action of these compounds. It is well known that the
liver plays a key role in the metabolism of orally administered
drugs.12 The rat liver microsome system is oen considered as
a reasonable model in which to study drug metabolism. On the
other hand, in vivo metabolic studies could comprehensively
reveal the metabolic pathways of drugs.
In recent years, the use of liquid chromatography coupled
with tandem mass spectrometry (LC-MS/MS) has been routinely
used to detect and identify metabolites13,14 and has been used to
study drug metabolism (avones and avonoids,15 phenyl-
propanoids,16–18 terpenes,19 alkaloids,20 saponins,21 stilbenes22
and traditional Chinese medicinal extracts23), pharmacoki-
netics and toxicokinetics of metabolites,24,25 and tissue distri-
bution and excretion of metabolites.26 In addition, this method
has applications in lipidomics,27 proteomics28 and metab-
olomics.29,30 A primary advantage of tandem mass spectrometry
(MS/MS) is the ability of this method to detect a broad range of
drugs with high sensitivity and specicity in a single analytical
run.31–34 In addition, high-resolution mass spectrometry
conrms structures by comparing the exact measured mass of
a compound with the exact theoretical mass.35–37
Fig. 1 Chemical structure of LQ.
Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical
University, Shijiazhuang 050017, P. R. China. E-mail: zhanglantong@263.net; Fax:
+86-311-86266419; Tel: +86-311-86266419
† Electronic supplementary information (ESI) available: Detailed information
regarding other metabolites found in vitro and in vivo. Fig. S1 MS/MS spectra of
all metabolites of LQ detected in vitro and in vivo. See DOI: 10.1039/c7ra13760e
To our knowledge, there has been one report of the meta-
bolic prole of LQ China;38 however, this report was incomplete
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and only identied 7 metabolites. In this research contribution, to 55, 55 and 35 L minꢂ1, respectively. The collision energy (CE)
a simple and rapid UHPLC-Q-TOF-MS/MS approach combined was set to ꢂ35 eV, and the collision energy spread (CES) was
with pattern recognition analysis was rst employed to rapidly 15 eV.
screen and characterize metabolites of LQ in vitro and in vivo,
Metabolite identication was performed with Metabo-
which was the rst systematic study of the metabolism of LQ in litePilot 1.5 (AB Sciex, CA, USA) based on accurate measure-
vitro and in vivo. The characterization of 56 in vivo metabolites ments of m/z values and on the processing of the data obtained
and 15 in vitro metabolites was achieved by UHPLC-Q-TOF-MS/ from the XIC (extracted ion chromatography), MDF (mass defect
MS analysis based on the MS/MS spectra and clog P values. In ltering), PIF (product ion ltering) and NLF (neutral loss
addition, the metabolic pathways of LQ were summarized. ltering) screening of putative metabolites. In addition,
These results provide insight into the metabolic mechanism of elemental compositions and chemical formulas were
LQ and lay the foundation for novel drug design.
calculated.
2. Materials and methods
2.3. Animals and drug administration
2.1. Chemicals and materials
Thirty male Sprague-Dawley (SD) rats, 10–12 weeks in age and
weighing 200–230 g, were obtained from the Experimental
Animal Center of Hebei (Shijiazhuang, China). Rats were
housed under standard temperature, humidity and light
conditions. The animals were kept in an environmentally
controlled breeding room for 7 days and fasted for 12 h before
experiments. LQ was dissolved in a 0.5% carboxymethyl cellu-
lose sodium (CMC-Na) aqueous solution. Thirty male SD rats
were divided into six groups of ve rats per group, which
included experimental blood, urine and feces, and bile groups
as well as blank blood, urine and feces, and bile groups. The
prepared LQ suspension was orally administered to 15 rats from
the experimental blood, urine and feces, and bile groups at
a dose of 120 mg kgꢂ1, and 0.5% CMC-Na aqueous solution was
orally administered to 15 rats from the blank blood, urine and
feces, and bile groups. All experiments were conducted in
accordance with the guides of Animal Care and Use Committee
at Hebei Medical University. This study was also performed in
LQ (CAS No: 551-15-5) was purchased from Chengdu Desert
Biotechnology Co., Ltd. (Chengdu, China). b-NADP (b-nicotin-
amine adenine dinucleotide phosphate), glucose-6-phosphate
(G-6-P) and glucose-6-phosphatedehydrogenase (G-6-PD) were
purchased from Sigma Chemical Co. (St. Louis, MO, USA).
MgCl2, UDPGA (uridine-50-diphosphoglucuronic acid trisodium
salt), Tris–HCl and alamethicin were purchased from BD
Biosciences (Woburn, MA, USA). Phosphate-buffered saline
(PBS) was purchased from Sangon Biotech Co., Ltd. (Shanghai,
China). HPLC-grade methanol was purchased from J.T. Baker
Chemical Company (Phillipsburg, NJ, USA). Formic acid (HPLC
grade) was provided by Diamond Technology (Dikma Technol-
ogies Inc., Lake Forest, CA, USA). Puried water was obtained
from Hangzhou Wahaha Group Co., Ltd. (Hangzhou, China).
2.2. Instrumentation and conditions
UHPLC-Q-TOF-MS/MS analysis was performed on a Shimadzu strict accordance with the NIH guidelines for the care and use of
UHPLC system (Shimadzu Corp., Kyoto, Japan) coupled with laboratory animals (NIH Publication No. 85-23 Rev. 1985) and
a triple TOF™ 5600+ MS/MS system (AB Sciex, CA, USA). was approved by the Institutional Animal Care and Use
Information-dependent acquisition (IDA) was carried out. Committee of National Tissue Engineering Center (Shanghai,
Chromatographic separation was conducted on a Poroshell 120 China).
EC-C18 (2.1 ꢀ 100 mm, 2.7 mm) column with a SecurityGuard®
Plasma sample (ve SD rats) collection was performed as
UHPLC C18 pre-column (Agilent Corp, Santa Clara, CA, USA). follows: blood was taken from the canthi of the rats 0.17, 0.50,
The column temperature was maintained at 25 ꢁC. The mobile 0.75, 1, 2, 4, 6, 9, 12 and 24 h aer administration. Aer
phase was consisted of water containing 0.1% formic acid (A) centrifugation at 1400 ꢀ g for 5 min (Hunan Xiangyi Laboratory
and methanol (B). The gradient elution program was optimized Instrument Development Co. Ltd., Hunan, China), the super-
for the separation, and the program was as follows: 0–2 min, 10– natant was collected, and all plasma samples were combined.
15% B; 2–15 min, 15–45% B; 15–20 min, 45–95% B; 20–25 min, Blank plasma was collected in the same manner from rats (ve)
95–95% B. Aer maintaining the column at 95% solvent B for administered 0.5% CMC-Na aqueous solution.
5 min, the column was returned to its starting conditions over
Urine and feces (ve SD rats) collection was performed as
1 min and was equilibrated in 10% solvent B for 5 min. The ow follows: urine and feces were collected during the 0–4 h, 4–8 h,
rate of the mobile phase was set to 0.3 mL minꢂ1, and the 8–12 h, 12–24 h, 24–36 h, 36–48 h, 48–60 h and 60–72 h periods
injection volume was 3 mL.
aer administration, and all the urine and feces samples were
A Triple TOF™ 5600 system with DuoSpray™ ion sources combined. Rats (ve) administered 0.5% CMC-Na aqueous
(AB Sciex Triple TOF™ 5600+, Concord, Ontario, Canada) solution were subjected to the same process to collect blank
operating in the negative electrospray ionization mode was used urine and feces samples.
for detection. The following MS/MS conditions were used: ion
Bile (ve SD rats) collection was performed as follows: rats
spray voltage, ꢂ4.5 kV; the turbo spray temperature, 550 ꢁC; and were administered urethane-containing physiological saline
declustering potential (DP), ꢂ60 V. Nitrogen was used as the solution (1.5–2 g kgꢂ1) aer gavage, and then, bile duct can-
nebulizer and auxiliary gas. Furthermore, the ows of the nulation. Then, bile samples were collected during 0–1 h, 1–3 h,
nebulizer gas (gas 1), heater gas (gas 2) and curtain gas were set 3–5 h, 5–8 h, 8–12 h, 12–20 h and 20–24 h periods aer
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administration. Finally, all bile samples were consolidated. Rats NADPH was added to initiate the reaction. Aer incubation for
(ve) administered 0.5% CMC-Na aqueous solution were sub- 30 min at 37 ꢁC in a metabolic shaker (1200 ꢀ g, Hangzhou Miu
jected to the same process to collect a blank bile sample.
Instruments Co., Ltd., Hangzhou, China), 1 mL of ice-cold ethyl
Three milliliters of blood, urine and bile samples were taken, acetate was added to stop the reaction, and the mixture was
and the protein in the samples was precipitated by methanol. vortexed for 5 min. Aer centrifugation at 10 000 ꢀ g for 10 min,
Then, the supernatant was concentrated to dryness under the organic phase was collected and evaporated under nitrogen
reduced pressure at 25 ꢁC using a Heidolph Laborota 4001 gas. Residues were redissolved in 100 mL of methanol, and an
rotatory evaporator (Heidolph Instruments, GmbH & Co., aliquot (3 mL) was injected into the chromatographic system for
Schwabach, Germany). The dried samples were dissolved in 300 analysis. The blank sample was incubated without LQ, while the
mL of methanol in an ultrasonic bath for 5 min, and then, the control sample was incubated without the NADPH-generating
samples were centrifuged for 10 min at 10 000 ꢀ g. Then, the system by following the method described above.
supernatant was injected into the UHPLC-Q-TOF-MS/MS system
for further analysis.
2.4.4. Phase II metabolism. The reaction mixture (total
volume 200 mL) containing 1.0 mg mLꢂ1 rat liver microsomes,
Methanol (20 mL) was added to the feces sample (2.0 g), and 2 mmol Lꢂ1 UDPGA, 8 mmol Lꢂ1 MgCl2, 25 mg mLꢂ1 alame-
then, the sample was ultrasonicated for 45 min (Kun Shan thicin in PBS (pH 7.4) and 100 mmol Lꢂ1 LQ methanol (nal
Ultrasonic Instruments Co., Kunshan, China). Aer the mixture amount in the reaction medium: 1%) solution was pre-
was centrifuged at 10 000 ꢀ g for 10 min, the supernatant was incubated for 20 min at 37 ꢁC, and then, UDPGA was added
collected and blow-dried in a nitrogen atmosphere. The residue to start the reaction. Aer incubation for 60 min at 37 ꢁC in
was dissolved in 400 mL of methanol and centrifuged at 10 000 a metabolic shaker (1200 ꢀ g), 200 mL of ice-cold methanol was
ꢀ g for 10 min. The supernatant (3 mL) was injected into the added to stop the reaction. Aer vortexing for 5 min, the organic
chromatographic instrument.
phase was collected and evaporated under nitrogen gas. Meth-
All the bio-samples were placed in the ꢂ80 ꢁC freezer for anol (100 mL) was added to the residues, and an aliquot (3 mL)
storage.
was injected into the chromatographic system for analysis. The
blank sample was incubated without LQ, while the control
sample was incubated without the UDPGA-generating system
following the method described above.
2.4. Microsomal incubation
2.4.1. Preparation of standard solutions. The appropriate
amount of standard LQ was accurately weighed and dissolved in
50% methanol to make the sꢁtandard solution (2.15 mg mLꢂ1).
The solution was stored at 4 C in a refrigerator.
3. Results and discussion
A total of 56 in vivo metabolites and 15 in vitro metabolites were
detected in the experimental conditions used. The metabolites
detected in vivo and in vitro are listed in Tables 1 and 2,
respectively, and the structures of the metabolites are shown in
Fig. 2. The XIC and MS/MS data of the metabolites detected in
vivo and in vitro are presented in Fig. 3 and S1,† respectively.
2.4.2. Preparation of liver microsomes. Liver microsomes
were prepared by differential centrifugation.39 All surgical
ꢁ
instruments and experimental reagents were stored at 4 C in
refrigerator. Five male SD rats (220–250 g) were fasted for 24 h
and decapitated. The liver was quickly retrieved, and the blood
was blotted with lter paper. Then, the liver was weighed and
was repeatedly washed with sugar solution till a khaki color.
Then, the liver was added to an ice-cold sugar solution that was
3.1. Analytical strategy and metabolite analysis
4 times the weight of the liver and was then cut and homoge- In this study, a method of metabolite identication was devel-
nized. Aer centrifugation at 20 000 ꢀ g for 20 min at 4 ꢁC, the oped that was based on a Triple TOF™ instrument with
precipitate was discarded. Aer additional centrifugation at a multiple mass defect lter (MMDF) combined with dynamic
100 000 ꢀ g for 60 min at 4 ꢁC, the supernatant was discarded. background subtraction (DBS)-dependent on-line data acquisi-
The precipitate was washed with 4 times as much cold Tris–HCl tion and multiple post-acquisition data mining technologies.
solution. Aer centrifugation at 100 000 ꢀ g for 60 min at 4 ꢁC, First, on-line data and accurate MS/MS data were acquired
the precipitation obtained was resuspended using 4 times as using a full-scan, unique and effective MMDF and a DBS-
much Tris–HCl solution to obtain liver microsomes. Finally, the dependent data acquisition method.41 Then, post-acquisition
liver microsomes were placed at ꢂ80 ꢁC in the freezer for data mining was performed using various data-mining tools
storage until further use. In addition, the protein concentration such as XIC, MDF, PIF and NLF.42 Furthermore, structures of
of the liver microsome suspension was determined by the Lowry the metabolites of LQ were elucidated based on accurate mass
method.40
measurements, knowledge of relevant drug bio-transformation,
2.4.3. Phase I metabolism. The typical incubation mixture previously investigated fragmentation patterns of LQ, and MS/
(200 mL nal volume) consisted of a 0.1 mol Lꢂ1 K2HPO4 buffer MS spectra of metabolites. Peakview 1.2 soware was used to
containing rat microsomes (1.0 mg mLꢂ1), 3.3 mmol Lꢂ1 MgCl2, identify possible metabolites by comparing the extracted ion
1.3 mmol Lꢂ1 b-NADPH, 3.3 mmol Lꢂ1 glucose-6-phosphate, 1.0 chromatograms and base peak chromatograms of the sample
U mLꢂ1 glucose-6-phosphate dehydrogenase and 100 mmol Lꢂ1 group with those of the blank group.43 Finally, the useful clog P
LQ methanol (nal amount in the reaction medium: 1%). Pre- parameter, which was calculated using the ChemDraw Ultra
incubation was carried out at 37 ꢁC for 5 min, and then, 12.0 program, was introduced to distinguish between structural
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isomers. Generally, compounds with the larger values have
longer retention times in reversed-phase liquid chromatog-
raphy systems.44,45
All chemical constituents in TMM can be categorized into
different families based on structural types. Thus, groups of
compounds with identical carbon skeletons may yield similar
fragmentation patterns and then generate the same character-
istic fragment ions when subjected to collision-induced disso-
ciation (CID) for mass spectrometry. Accordingly, a core
supplementary tool in this approach is to use key product ions
(KPIs) as markers for compounds detection and identica-
tion.46–48 In this study, a KPI at m/z 255.0651 could be generated
from common substructures as standards and was selected as
a diagnostic ion for detecting relevant analogues of these
substructures (shown in Fig. 3).
3.2. Mass fragmentation behavior of LQ
The retention time of LQ was 12.01 min, and LQ produced
a molecular ion [M ꢂ H]ꢂ at m/z 417.1176 under the experi-
mental conditions. Moreover, major secondary fragment ions at
m/z 255.0651 [M–H–C6H10O5]ꢂ, 135.0079 (RDA reaction (retro
DielsꢂAlder reaction)), 119.0496 (RDA reaction) and 91.0190
(RDA reaction) were detected. The proposed MS/MS fragmen-
tation behavior and fragmentation pathways of LQ are shown in
Fig. 4. The molecular weight and elemental composition of LQ
were used as a baseline for comparison with some metabolites.
3.3. Identication of in vivo phase I metabolites
3.3.1. Oxidation reaction
Metabolites M1, M2 and M3. M1–M3 were eluted at 7.95, 9.54
and 13.20 min, respectively, with deprotonated molecular ions
[M ꢂ H]ꢂ at m/z 319.0477, 319.0477 and 319.0482, respectively,
which were 98 Da less than the value obtained for LQ and cor-
responded to C15H12O8. The fragment ions at m/z 273.1696,
255.1595, 239.0923, 221.0814 and 151.0475 were produced from
M1, M2 and M3 by the loss of CO, H2O, O and by RDA reaction.
Among these fragments, the fragment ion at m/z 255.1595 was
hydrolyzed LQ. M1, M2 and M3 were oxidation metabolites of
LQ and were assigned based on clog P values of 0.509837,
0.569225 and 0.629837 by ChemDraw 12.0 soware.
Metabolites M7, M8, M9 and M10. M7–M10, eluted at 9.01,
12.61, 15.65 and 16.45 min, respectively, were characterized as
deprotonated molecular ions [M ꢂ H]ꢂ at m/z 433.1115,
433.1111, 433.1115 and 433.1114, respectively, which were
16 Da more than the value obtained for the parent drug LQ. This
nding suggested that M7–M10 were oxidation metabolites of
LQ and that the chemical formula of M7–M10 was C21H22O10.
The observed diagnostic fragment ions at m/z 271.0596,
243.0645, 227.0692, 136.0151 and 109.0290 were generated by
the loss of C6H10O5, CO and O and by RDA reaction. The clog P
values of M7–M10 were 0.300687, 0.500687, 0.558399 and
0.948399, respectively. Therefore, the four compounds were
immediately identied on the basis of the retention times and
clog P values.
Metabolites M11, M12, M13 and M14. M11–M14 were eluted at
9.11, 10.84, 11.67 and 13.12 min, respectively, with
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Table 2 Summary of phase I and phase II metabolites of LQ in vitroa
Metabolites Composition
Error tR
Score
ID
shi
Formula
m/z
(ppm) (min) (%)
MS/MS fragments
clog P
Blanks Controls Samples
N1
N2
N3
N4
N5
Oxidation
Oxidation
Oxidation
Oxidation
Hydrolysis +
oxidation
C21H22O10 433.1110 ꢂ2.0
8.62 79.1 271.0592, 243.0645, 109.0289 0.300687
9.01 79.0 271.0595, 243.0647, 109.0282 0.500687
9.51 82.5 271.0596, 243.0645, 109.0292 0.558399
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
+
+
+
+
+
C
21H22O10 433.1111 ꢂ2.8
21H22O10 433.1112 ꢂ2.6
C
C21H22O10 433.1109 ꢂ2.2 14.03 84.4 271.0589, 243.0676, 109.0290 0.948399
C15H12O5 271.0596 ꢂ1.1 11.12 89.7 253.0474, 151.0016, 135.0439, 1.93694
119.0487, 91.0191
N6
N7
N8
Hydrolysis +
oxidation
Hydrolysis +
oxidation
C
15H12O5 271.0595 ꢂ1.3 13.60 81.1 253.0486, 151.0024, 135.0444, 2.05485
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
+
+
+
119.0495, 91.0136
C15H12O5 271.0592 ꢂ1.5 13.83 73.4 253.0480, 151.0021, 135.0435, 2.44485
119.0480, 91.0131
Desaturation C21H20O9 415.1011 ꢂ1.7 12.63 76.2 253.0518, 252.0421, 223.0455, 1.06205
142.99500, 112.9840
N9
N10
Hydrolysis
C
15H12O4 255.0650 ꢂ3.4 16.03 96.8 135.0083, 119.0500, 91.0193
2.53394
2.90529
ꢂ
ꢂ
ꢂ
ꢂ
+
+
Hydrolysis +
desaturation +
oxidation
C15H10O5 269.0437 ꢂ3.9 16.38 79.2 135.0090, 133.0287
N11
Hydrolysis +
desaturation
Glucuronide
conjugation
Hydrolysis +
glucuronide
conjugation
Hydrolysis +
glucuronide
conjugation
Glucose
C
15H10O4 253.0496 ꢂ2.4 17.58 85.6 135.0077, 133.0284,
2.5753
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
ꢂ
+
+
+
117.0341, 91.0187
N12a
N12b
N13
C27H30O15 593.1565 3.7
7.62 74.4 417.1208, 255.0659, 175.0241, ꢂ1.22216
135.0078, 117.0187, 113.0241 ꢂ1.01791
11.19 86.4 255.0662, 226.9648, 175.0239, 0.27429
135.0080, 119.0495, 113.0242
C
21H20O10 431.0989 1.3
21H20O10 431.0985 0.2
N14
C
11.93 82.8 255.0666, 226.9703, 175.0233, 0.55864
135.0079, 119.0496, 113.0229
ꢂ
ꢂ
ꢂ
ꢂ
+
+
N15
C27H32O14 579.1752 3.9
12.45 89.3 417.1273, 402.9939, 255.0669, ꢂ0.743309
conjuagtion
238.9306, 135.0085, 119.0492
a
+ Detected, ꢂ undetected, a and b – possible metabolites.
deprotonated molecular ions [M ꢂ H]ꢂ at m/z 447.0909,
447.0891, 447.0903 and 447.0909, respectively, which were
30 Da more than the value obtained for LQ. The major fragment
ions detected at m/z 271.0600, 135.0443 and 113.0242 were
generated by the loss of C6H8O6 and by RDA reaction, which
implied that M11–M14 were oxidation products of LQ and that
the chemical formula was C21H20O11. In addition, M11, M12,
M13 and M14 were assigned based on the clog P values of M11,
M12, M13 and M14, which were ꢂ0.178159, 0.0218411,
0.0795532 and 0.469553, respectively.
Metabolite M18. M18 was detected at a retention time of
12.89 min, with a deprotonated molecular ion [M ꢂ H]ꢂ at m/z
301.1284, 46 Da higher than that of M0 losing C6H10O5, which
suggested that M18 was oxidation metabolite. The fragment
ions at m/z 283.1184, 255.0857, 239.1275, 169.0858 and
118.9799 were gained by loss of H2O, CO, O and by RDA reac-
tion. According to the fragmentation, the chemical formula of
M18 was C15H10O7.
Metabolite M20. M20 had a retention time of 16.21 min and
was detected at m/z 285.0791 ([M ꢂ H]ꢂ), which was 30 Da more
than the value obtained for hydrolyzed LQ, which implied that
oxidation and methylation reactions had occurred. Represen-
tative fragment ions were observed at m/z 255.0729 and
119.0495 due to loss of CH2O and due to RDA reaction, which
suggested that the formula of M20 was C16H14O5.
Metabolite M23. A peak was eluted at a retention time of
17.03 min. The MS/MS spectrum of M23 showed a deprotonated
molecular ion [M ꢂ H]ꢂ at m/z 415.1780, which was 2 Da less
than the value obtained for M0, which conrmed the molecular
formula to be C21H20O9. In addition, the MS/MS spectrum of
M23 showed a number of characteristic fragment ions at m/z
253.0132 [M–C6H10O5–H]ꢂ, 142.9952 (RDA reaction) and
112.9901 (RDA reaction).
3.3.2. Reduction reaction
Metabolite M24. M24 was detected at 17.58 min and showed
a deprotonated molecular ion [M ꢂ H]ꢂ at m/z 253.0509, which
was 164 Da lower than the value obtained for M0, which sug-
gested that the loss of two hydrogens from hydrolyzed M0.
Meanwhile, representative fragment ions at m/z 135.0082,
133.0291 and 117.0346 were obtained due to RDA reaction. The
formula was identied as C15H10O4.38
Metabolite M19. M19 was detected at a retention time of
14.64 min with a deprotonated molecular ion [M ꢂ H]ꢂ at m/z
269.0464, which was generated by the loss of two oxygens from
M18. Typical fragment ions at m/z 241.0512, 225.0547, 135.0085
and 133.0286 were detected due to successive loss of CO and O
and due to RDA reaction. Thus, M19 was deduced to be
3.3.3. Hydrolysis reaction
C
15H10O5.
Metabolites M25, M26. M25 and M26 displayed molecular
ions [M ꢂ H]ꢂ at m/z 269.0780 and 269.0781, respectively, and
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Fig. 2 Chemical structures of all metabolites of LQ detected in vitro and in vivo (a, b and c—possible chemical structure).
had retention times of 12.40 and 13.87 min, respectively. The
masses of the deprotonated M25 and M26 were 14 Da higher
than the mass of hydrolyzed LQ, which suggested that a meth-
ylation reaction occurred on LQ aer the loss of C6H10O5. The
MS/MS spectra showed a characteristic fragment ion at m/z
253.0637 [M–O–H]ꢂ, which implied that the formula of M25 and
M26 was C16H14O4. Then, M25 and M26 were identied based
on their clog P values of 2.83559 and 3.11994, respectively.
Metabolite M27. The metabolite M27 exhibited a deproto-
nated ion [M ꢂ H]ꢂ at m/z 255.0661, which was 162 Da lower
than the value obtained for M0, conrming the occurrence of
hydrolysis. In addition, typical fragment ions at m/z 135.0090,
119.0510 and 91.0204 were produced by RDA reaction. The
formula of M27 was deduced to be C15H12O4.38
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Fig. 3 Extracted ion chromatograms of all metabolites of LQ detected in vitro and in vivo. (A1–2 in rat blood sample, B1–7 in rat urine sample, C
in rat bile sample, D in rat feces sample, E1–4 in rat liver microsomes). Additional graph: metabolites detected by KPIs in rat blood, urine, bile and
feces samples.
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Fig. 3 (Contd.)
M0. The product ions detected at m/z 447.0917, 271.0599,
175.0237, 135.0081 and 113.0242 were produced by the loss of
SO3 and C6H8O6 and by RDA reaction, which suggested that the
formula was C21H20O14S. In addition, the clog P values of M29,
M30 and M31 were ꢂ1.91668, ꢂ1.61368 and ꢂ1.15368,
respectively, and M29, M30 and M31 were identied based on
this information.
3.4.3. Metabolites M32, M33. Two peaks were eluted at 7.63
and 12.01 min under the chromatography conditions used. M32
and M33 were isomers and had identical MS/MS spectra. M32
and M33 had deprotonated molecular ions [M ꢂ H]ꢂ at m/z
593.1479 and 593.1476, respectively, which were 176 Da higher
than the value obtained for M0, suggesting that M32 and M33
were glucuronide conjugation metabolites. Moreover, the MS/
3.4. Identication of in vivo phase II metabolites
3.4.1. Metabolite M28. A peak was detected at 7.33 min and
exhibited a deprotonated molecular ion [M ꢂ H]ꢂ at m/z
607.1310, which was 190 Da higher than the value obtained for
LQ. Secondary fragment ions were detected at m/z 431.0975 [M–
C6H8O6–H]ꢂ and 255.0655 [M–2C6H8O6–H]ꢂ, suggesting that
hydrolyzed LQ had undergone bis-glucuronide conjugation. In
addition, the chemical formula was deduced to be C27H28O16.
3.4.2. Metabolites M29, M30, M31. M29–M31 were identi-
ed as sulfate and glucuronide conjugation metabolites. M29–
M31 were eluted at 7.55, 8.32 and 10.20 min and had deproto-
nated molecular ions [M ꢂ H]ꢂ at m/z 527.0471, 527.0471 and
527.0480, which were 110 Da more than the value obtained for
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Fig. 4 MS/MS spectrum of LQ and its predominant fragmentation pathways.
MS spectra showed fragment ions at m/z 417.1143, 255.0658, 119.0503 were observed due to the loss of C6H8O6 and due to
135.0101 and 119.0517 due to the loss of C6H8O6 and C6H10O5 RDA reaction. It was evident that hydrolyzed LQ had undergone
and due to RDA reaction. In addition, the formula was deduced glucuronide conjugation, and the molecular formula was
to be C27H30O15
.
38 Then, M32 and M33 were identied based on identied to be C21H20O10
.
38 M44 and M45 were eluted at 11.17
their clog P values of ꢂ1.22216 and ꢂ1.01791, respectively.
and 11.97 min, respectively. The clog P values of M44 and M45
3.4.4. Metabolite M34. M34 was eluted at 8.05 min with were 0.27429 and 0.55864, respectively. Hence, M44 and M45
a deprotonated molecular ion [M ꢂ H]ꢂ at m/z 415.1517, which were identied on the basis of their retention times and clog P
was 2 Da less than the value obtained for the parent drug M0. values.
Secondary fragment ions at m/z 335.0230 [M–SO3–H]ꢂ and
3.4.8. Metabolites M46, M47. The metabolites M46 and
255.0658 [M–2SO3–H]ꢂ were detected in the MS/MS spectrum, M47, eluted at 12.13 and 12.78 min, respectively, were observed
suggesting that hydrolyzed LQ had undergone a bis-sulfate in the metabolic proles with deprotonated molecular ions [M
conjugation reaction and that the formula was C15H12O10S2.
ꢂ H]ꢂ at m/z 429.0828 and 429.0824, respectively, correspond-
3.4.5. Metabolite M35. M35 had a retention time of ing to an elemental composition of C21H18O10. The masses of
8.70 min with a deprotonated molecular ion [M ꢂ H]ꢂ at m/z the deprotonated M46 and M47 were 12 Da more than the mass
497.0759, which was 80 Da higher than the value obtained for of LQ. A characteristic fragment ion at m/z 253.0505 [M–
LQ, suggesting that M35 was a metabolite of sulfate conjuga- C6H8O6–H]ꢂ was observed, which conrmed that M46 and M47
tion. Meanwhile, characteristic fragment ions at m/z 417.1185 were metabolites of glucuronide conjugation. Moreover, M46
[M–SO3–H]ꢂ, 255.0655 [M–SO3–C6H10O5–H]ꢂ and 135.0074 and M47 were assigned based on clog P values of 0.390001 and
(RDA reaction) were seen in the MS/MS spectrum, implying that 0.583201, respectively.
the chemical formula was C21H22O12S.38
3.4.9. Metabolites M50, M51. M50 and M51 showed
3.4.6. Metabolite M36. M36 exhibited a deprotonated deprotonated molecular ions [M ꢂ H]ꢂ at m/z 335.0216 and
molecular ion [M ꢂ H]ꢂ at m/z 511.0541, which was 94 Da more 335.0217, respectively, which were 82 Da less than the value
than the value obtained for deprotonated LQ. In addition obtained for LQ. M50 and M51 were eluted at 12.49 and
a series of important fragment ions were observed at m/z 17.30 min, respectively. Typical fragment ions at m/z 255.0656
431.0970 [M–SO3–H]ꢂ, 255.0654 [M–SO3–C6H8O6–H]ꢂ, 135.0084 [M–SO3–H]ꢂ, 135.0083 (RDA reaction) and 119.0500 (RDA
(RDA reaction) and 119.0506 (RDA reaction) in the MS/MS scan. reaction) were observed in the MS/MS spectra. The neutral loss
Moreover, the retention time of M36 was 8.92 min, and the of SO3 suggested that hydrolyzed LQ had undergone sulfate
formula was identied to be C21H20O13S.38
conjugation, implying that the molecular formula was
C
15H12O7S.38 By using ChemDraw 12.0, the clog P values of M50
3.4.7. Metabolites M44, M45. The QTOF-MS mass spectra
of the M44 and M45 metabolites exhibited deprotonated ions and M51 were 0.689589 and 0.97394, respectively. Hence, these
[M ꢂ H]ꢂ at m/z 431.0976 and 431.0976, respectively, which metabolites were identied based on the retention times and
were 14 Da higher than the value obtained for the parent drug clog P values.
LQ. In addition, both metabolites exhibited the loss of a glucu-
3.4.10. Metabolites M53, M54. Two peaks were eluted at
ronide unit (176 Da) in the MS/MS spectra. A number of typical 13.24 and 14.22 min with deprotonated molecular ions [M ꢂ H]ꢂ
secondary fragment ions at m/z 255.0658, 135.0086 and at m/z 413.0854 and 413.0853, respectively, which were 4 Da less
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Fig. 5 Metabolic profile and proposed metabolic pathways of LQ in vitro and in vivo (a, b, c—possible chemical structure).
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Fig. 5 (Contd.)
than the value obtained for the parent compound LQ. These deprotonated molecular ions [M ꢂ H]ꢂ at m/z 433.1110,
metabolites had identical fragment ions at m/z 255.0666, 135.0087 433.1111, 433.1112 and 433.1109, respectively, which were
and 119.0501. The fragmentation ion at m/z 255.0666 suggested 16 Da more than the value obtained for LQ, suggesting that N1–
that the position of oxygen loss was glucose and not avone. N4 were oxidation metabolites and corresponded to the
Therefore, the molecular formula was deduced to be C21H18O9. In molecular formula C21H22O10. In addition, typical fragment
addition, metabolites M53 and M54 were identied based on the ions at m/z 271.0592, 243.0645 and 109.0289 were generated by
clog P values of 0.29614and 0.34904, respectively.
the loss of C6H10O5 and CO and by RDA reaction. Moreover, N1–
3.4.11. Metabolite M55. M55 was eluted at a retention time N4 were identied based on the clog P values of N1–N4 at
of 16.56 min with a deprotonated molecular ion [M ꢂ H]ꢂ at m/z 0.300687, 0.500687, 0.558399 and 0.948399, respectively.
459.1297, which was 42 Da more than the value obtained for LQ.
Metabolite N8. N8 was detected at 12.63 min and exhibited
Masses for characteristic ions were observed at m/z 417.1198, a sharp peak for a deprotonated molecular ion [M ꢂ H]ꢂ at m/z
255.0645, 135.0070 and 119.0489, which were generated by the 415.1011, which was 2 Da less than the value obtained for M0.
loss of CH2CO and C6H10O5 and by RDA reaction, suggesting Fragment ions at m/z 253.0518 [M–C6H10O5–H]ꢂ suggested that
that M55 was an acetylation metabolite. Moreover, the chemical a desaturation reaction had occurred, which was consistent
formula of M55 was C23H24O10
.
with a molecular formula of C21H20O9.
3.4.12. Metabolite M56. M56 eluted at 18.33 min and had
Metabolite N9. N9 was detected at 16.03 min. N9 exhibited
a deprotonated ion [M ꢂ H]ꢂ at m/z 431.0992, which was 14 Da a sharp peak for a deprotonated molecular ion [M ꢂ H]ꢂ at m/z
higher than the value obtained for M0, implying that methyla- 255.0650, which was 162 Da lower than the value obtained for
tion had occurred. The secondary fragment ions at m/z 255.0666 LQ, implying that a hydrolysis had occurred. The secondary
[M–CH2–C6H10O5–H]ꢂ and 135.0086 (RDA reaction) demon- fragment ions at m/z 135.0083 and 119.0500, caused by RDA
strated that the chemical formula was C22H24O9. Other metab- reaction, conrmed that the molecular formula of N9 was
olites were showed in ESI.†
C
15H12O4.38
Metabolite N10. N10 exhibited a deprotonated molecular ion
[M ꢂ H]ꢂ at m/z 269.0437, which was 14 Da more than the value
obtained for N9, indicating that the molecular formula of N10
was C15H10O5. In addition, N10 had a retention time of
16.38 min, and the important fragment ions were at m/z
135.0090 (RDA reaction) and 133.0287 (RDA reaction).
3.5. Identication and characterization of in vitro
metabolites
In this incubation system, een metabolites were detected,
including eleven phase I and four phase II metabolites. Further-
more, metabolites N1–N14 were identied in the in vivo metabolic
study. However, N15 was not found in vivo bio-samples. The
structures of all the metabolites are shown in Fig. 2.
3.5.1. Identication of phase I metabolites
Metabolites N1, N2, M3 and N4. N1–N4, with retention times
of 8.62, 9.01, 9.51 and 14.03 min, exhibited sharp peaks of
Metabolite N11. N11 showed a deprotonated molecular ion [M
ꢂ H]ꢂ at m/z 253.0496, which was 2 Da less than the value ob-
tained for N9, suggesting that a desaturation reaction occurred
for N9, and the molecular formula of N11 was predicted to be
C
15H10O4.38 In addition, the secondary fragment ions at m/z
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135.0077, 133.0284 and 117.0341, caused by RDA reaction, were
consistent with the chemical structure of N11. Moreover, the
retention time of N11 was 17.58 min.
3.8. Comparison of metabolites identied in this study and
in a previous study
A study of the metabolic pathways of LQ has been reported in
China, which only detected 7 metabolites (M24, M27, M32
(N12), M35, M36, M44 and M45 (N13 and N14), and M51).
However, in this study, 56 metabolites were detected in vivo and
15 metabolites were detected in vitro, including the 7 metabo-
lites identied in the previous study. Moreover, the metabolic
sites of two metabolites were not determined in the previous
study, and there were some differences between this study and
the previous study. In this manuscript, M32, M33 and M50, M51
were detected as two pairs of isomers; however, these metabo-
lites were detected as a single chromatographic peak in the
previous study. Moreover, in this study, M32 and M33 were both
not detected in vitro (N12), and no isomers of M36 were detected
in vivo. Furthermore, in this manuscript, the metabolic sites of
M44 and M45 were identied based on the important clog P
values, which were the same as those of N13 and N14 in vitro. In
addition, in this study, metabolite N15 was not detected in vivo;
therefore, it was speculated that N15 had undergone oxidation
and that metabolites M32 and M33 were found in vivo.
3.5.2. Identication of phase II metabolites
Metabolite N12. N12 presented a deprotonated molecular ion
[M ꢂ H]ꢂ at m/z 593.1565, which was 176 Da higher than the
value obtained for LQ, suggesting that glucuronide conjugation
had occurred. In addition, the molecular formula was deter-
mined to be C27H30O15.38 Moreover, the MS/MS spectrum
exhibited a series of typical fragment ions at m/z 417.1208 [M–
C6H8O6–H]ꢂ, 255.0659 [M–C6H8O6–C6H10O5–H]ꢂ, 135.0078
(RDA reaction) and 117.0187 (RDA reaction). In addition, N12
was eluted at 7.62 min.
Metabolite N15. The molecular formula was determined to be
C
27H32O14 based on the deprotonated molecular ion [M ꢂ H]ꢂ
at m/z 579.1752, which was 162 Da higher than the value ob-
tained for LQ. N15 was eluted at 12.45 min and had character-
istic fragment ions at m/z 417.1273 [M–C6H10O5–H]ꢂ, 255.0669
[M–2C6H10O5–H]ꢂ, 135.0085 (RDA reaction) and 119.0492 (RDA
reaction), which implied that N15 was a glucose metabolite.
Other metabolites were showed in ESI.†
3.6. Metabolic pathways of LQ
4. Conclusion
Based on the elemental compositions of the metabolites, the
accurate MS/MS spectra, the chemical structures of the
metabolites and the fragment ions of the metabolites, the
metabolic pathway of LQ could be tentatively proposed. In total,
56 in vivo metabolites and 15 in vitro metabolites were charac-
terized. Based on these results, LQ mainly underwent oxidation,
reduction, hydrolysis, methylation, acetylation, glucuronide
and sulfate conjugation. Based on the in vivo metabolic data,
sulfate and glucuronide conjugation reactions were the major
bio-transformations. However, based on the in vitro metabolic
data, the metabolic pathways of LQ were concentrated in phase
I. The proposed metabolic pathways of LQ in vitro and in vivo are
shown in Fig. 5.
In conclusion, the UHPLC-Q-TOF-MS/MS method was rst
established to screen and identify the metabolites of LQ in vitro
and in vivo. Based on the preceding data acquisition and mining
strategy, twenty, y-four, forty-eight, thirty-seven and een
metabolites were detected in rat blood, urine, bile, feces and
liver microsomes, respectively. The metabolic pathways of LQ
were determined to be oxidation, reduction, hydrolysis, meth-
ylation, acetylation, and sulfate and glucuronide conjugation;
these ndings lled in the gaps remaining from the previous
incomplete study. Key product ion (KPI) was used to aid the
detection of metabolites of LQ. The results lay the foundation
for active screening studies. In addition, this study demon-
strated a powerful strategy for rapid screening and identifying
metabolites and chemical constituents of traditional Chinese
medicines.
3.7. Comparison of metabolic pathways in vitro and in vivo
Drug metabolism plays an important role in different areas of
the pharmaceutical industry and in drug development and
toxicology. The in vivo approach is quantitative and very effec-
tive in studies of drug metabolism.49,50 The in vitro method is
generally suitable for targeted studies and is oen predictive of
real hazards and risks.51 In this study, in vitro (rat liver micro-
somes) and in vivo (blood, urine, feces and bile in rats) meta-
bolic proles were investigated by UHPLC-Q-TOF-MS/MS.
Consequently, a total of 56 in vivo metabolites and 15 in vitro
metabolites were screened and characterized. The in vitro and in
vivo metabolisms were concentrated in phase I and II, respec-
tively. The metabolites detected in vitro were all detected in vivo
as well. However, the isomer of N12 was detected in vivo, and
N15 was not found in vivo. It was speculated that oxidation of
N15 had occurred in vivo. Therefore, the isomer of N12 was
observed in vivo, which suggested that the metabolism in vivo
was more complex and elusive than that in vitro.
Conflicts of interest
All the authors have declared no conict of interest.
Acknowledgements
The work received nancial support from the National Natural
Science Foundation of China (No. 81473180).
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