Effect of weakly basic conditions on the separation and purification of flavonoids and glycosides from chrysanthemum morifolium tea

Article abstract of DOI:10.3390/molecules24020297

Tea brewed from chrysanthemum flowers has been widely used in Chinese medicine. The possibly medicinal compounds in Chrysanthemum morifolium tea can be purified by preparative high performance liquid chromatography (HPLC), but this is usually done with acidic conditions, which leads to the hydrolysis of glycosides. In hopes of avoiding this hydrolysis, we explored the effect of weakly basic conditions on the separation and purification of flavonoids and glycosides from Chrysanthemum morifolium. We also explored the effects of weakly basic conditions on chlorogenic acid (3-CQA) and apigenin-7-O-glucoside (A7G). Our results show that the concentration of the weakly basic ammonium hydrogen carbonate and time had no significant effect on A7G, p < 0.01, but it had a significant effect on 3-CQA, p < 0.01. HPLC and ultraviolet (UV) analysis showed that the structure of 3-CQA is destroyed in weakly basic conditions. Caffeic acid, quinic acid, and 3,4-dihydroxymandelic acid, which is a hydrolysate of 3-CQA, were identified by ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The results showed that weakly basic conditions could be used for the purification of flavonoids and glycosides but not for caffeoylquinic acids. Moreover, our work clarified the hydrolysis behaviour of caffeoylquinic acids, which can be helpful for research into their functional aspects.

Full text of DOI:10.3390/molecules24020297

molecules
Article
Effect of Weakly Basic Conditions on the Separation
and Purification of Flavonoids and Glycosides from
Chrysanthemum morifolium Tea
Yuxiao Wang 1,3,4, Zhenzhen Xu 2, Yue Wu 1,3,4, Mo Li 1,3,4, Sicheng Pang 1,3,4, Zhe Liang 1,3,4 and
Yuanying Ni 1,3,4,
*
1
College of Food Science and Nutritional Engineering, China Agricultural University, Beijing 100083, China;
wangg5211@163.com (Y.W.); wy1102742656@163.com (Y.W.); limo_0125@163.com (M.L.);
13031162533@163.com (S.P.); liangzhe@cau.edu.cn (Z.L.)
Institute of Quality Standard & Testing Technology for Agro-Products, Chinese Academy of Agricultural
Sciences, Beijing 100083, China; xuzhenzhen@caas.cn
National Engineering Research Center for Fruit and Vegetable Processing, Beijing 100083, China
Key Laboratory of Fruit and Vegetable Processing, Ministry of Agriculture, Beijing 100083, China
Correspondence: niyycau@hotmail.com; Tel: +86-010-62737514
2
3
4
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Mingfu Wang and Yueliang Zhao
Received: 30 December 2018; Accepted: 12 January 2019; Published: 15 January 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Tea brewed from chrysanthemum flowers has been widely used in Chinese medicine.
The possibly medicinal compounds in Chrysanthemum morifolium tea can be purified by preparative
high performance liquid chromatography (HPLC), but this is usually done with acidic conditions,
which leads to the hydrolysis of glycosides. In hopes of avoiding this hydrolysis, we explored the
effect of weakly basic conditions on the separation and purification of flavonoids and glycosides from
Chrysanthemum morifolium. We also explored the effects of weakly basic conditions on chlorogenic acid
(3-CQA) and apigenin-7-O-glucoside (A7G). Our results show that the concentration of the weakly
basic ammonium hydrogen carbonate and time had no significant effect on A7G, p < 0.01, but it had a
significant effect on 3-CQA, p < 0.01. HPLC and ultraviolet (UV) analysis showed that the structure of
3-CQA is destroyed in weakly basic conditions. Caffeic acid, quinic acid, and 3,4-dihydroxymandelic
acid, which is a hydrolysate of 3-CQA, were identified by ultra-performance liquid chromatography
quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS). The results showed that weakly basic
conditions could be used for the purification of flavonoids and glycosides but not for caffeoylquinic
acids. Moreover, our work clarified the hydrolysis behaviour of caffeoylquinic acids, which can be
helpful for research into their functional aspects.
Keywords:
weakly basic condition; Chrysanthemum morifolium tea; chlorogenic acid;
apigenin-7-O-glucoside; hydrolysis
1. Introduction
Tea brewed from chrysanthemum flowers has been extensively used for centuries to treat a
variety of medical conditions, including chest pain, high blood pressure, diabetes, and headaches.
The benefits of Chrysanthemum morifolium are closely related to the composition and content
of phenolic compounds [
have been investigated [
isolated from Chrysanthemum morifolium flowers and related species by high performance thin-layer
chromatography (HPTLC). Uehara [ ] isolated five flavonoid glycosides (luteolin-7-O-glucoside
and 7-O-glucuronides of luteolin, apigenin, eriodictyol, and naringenin) from chrysanthemum
1
–
3]. Many functional ingredients from Chrysanthemum morifolium tea
3–
8
]. Guzelmeric [ ] determined the fingerprint of apigenin-7-O-glucoside
3
4
species by HPLC with a photodiode array (PDA). Lin [
5] identified 15 flavonoids and 15 caffeic
Molecules 2019, 24, 297; doi:10.3390/molecules24020297
www.mdpi.com/journal/molecules

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acid derivatives isolated from Chrysanthemum morifolium by liquid chromatography with a diode
array and characterised by electrospray ionisation/mass spectrometry (LC–DAD–ESI/MS). Han [6]
identified apigenin-7-O-600-malonyl-glucoside, luteolin-7-O-rutinoside, quercetin-7-O-galactoside,
quercetin-3-O-glucoside, apigenin-7-O-rutinoside, and chlorogenic acid from Chrysanthemum
morifolium species by ultra-performance liquid chromatography quadrupole time-of-flight mass
spectrometry (UPLC-Q-TOF-MS). Yuan [
9] analysed chlorogenic acid, luteolin-7-O-β-d-glucoside,
3,5-di-caffeoylquinic acid, apigenin-7- -d-glucopyranoside, and luteolin from chrysanthemum flowers
β
by HPLC-DAD. These studies showed that chrysanthemum flowers contain caffeoylquinic acids,
flavonoids, and glycosides.
Sensitive and efficient methods for the purification of these functional compounds from
chrysanthemums are scarce in the scientific literature. The most common methods for separation and
purification have been column chromatography, preparative high-performance liquid chromatography
(Pre HPLC) [10], high-speed countercurrent chromatography (HSCCC) [11], and thin-layer
chromatography (TLC) [12]. Unlike other methods, Pre-HPLC can isolate the many components of
Chrysanthemum morifolium tea simultaneously, but the acid used for improving peak performance
would hydrolyse the glycosides.
It is unknown whether weakly basic conditions will help to purify and analyse the components
of Chrysanthemum morifolium, thus we explored this possibility by: (1) analysis of the components
isolated in weakly basic conditions by HPLC-DAD; and (2) investigation of the hydrolytic behaviour
of chlorogenic acid (3-CQA) and apigenin-7-O-glucoside (A7G) with UPLC-Q-TOF-MS. We used
ammonium hydrogen carbonate to provide the weakly basic conditions because it decomposes above
36 ◦C to form ammonia, carbon dioxide, and water, which are easily removed from solvent systems.
This study provides an effective approach for isolating flavonoids and glycosides from Chrysanthemum
morifolium and clarifies the hydrolysis mechanism of caffeoylquinic acids, which will be helpful for
research on the purification and analysis of related substances.
2. Results and Discussion
2.1. Analysing the Components of Chrysanthemum in Weakly Basic Conditions
Figure 1a shows the components that can be isolated from Chrysanthemum morifolium in optimised
acidic conditions. The components that were previously identified by Q-TOF-MS [10] mainly include
chlorogenic acid (3-CQA), luteolin-7-O-glucoside, 3,5-dicaffeoylquinic acid, apigenin-7-O-glucoside
(A7G), apigenin-7-O-glucuronide, luteolin-7-O-600-malonylglucoside, and apigenin. Figure 1a shows
that these compounds are in lower concentrations in C. morifolium ‘Gongju’ and ‘Boju’ than for other
cultivars. The concentrations of 3-CQA and 3,5-dicaffeoylquinic acid in C. morifolium ‘Taiju’ are higher
than for other cultivars. There are much higher concentrations of A7G and apigenin in C. morifolium
‘Huangju’. The introduction of formic acid increased the retention and separation effect in the
purification process by preparative HPLC, but in the acidic conditions, the glycosides hydrolysed [10].

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Figure 1. HPLC analysis of the components from Chrysanthemum morifolium tea for acidic and basic
conditions. The gradient elution solvents consisted of phase A and phase B (acetonitrile). ( ) Phase A was
water with 0.1% formic acid, and ( ) phase B was water with 10 mmol/L ammonium hydrogen carbonate.
a
b
GJ, C. morifolium ‘Gongju’; HBJ, C. morifolium ‘Hangbaiju’; TJ, C. morifolium ‘Taiju’; BJ, C. morifolium
‘Boju’; CJ, C. morifolium ‘Chuju’; HJ, C. morifolium ‘Huangju’.
We investigated the effect of weakly basic conditions on the separation of the components from
chrysanthemum flowers (see Figure 1b). Most reversed phase C18 columns require acidic conditions
rather than basic conditions, but they can be used in basic conditions with different packing materials or
bonding methods. We used a C18 column (Athena C18-WP, 250
×
4.6 mm i.d., 5 µm, Anpel Laboratory
Technologies Inc., Shanghai, China) that works in basic conditions. Because ammonium hydrogen
carbonate (NH4HCO3) solutions are weakly basic, and because NH4HCO3 decomposes into the easily
removed NH3, CO2, and H2O, we chose it to generate the weakly basic conditions. The gradient elution
solvents that consisted of phase A (water with 10 mmol/L NH4HCO3) and phase B (acetonitrile) were
optimised as follows: from 0 to 10 min with 10–10% B, from 10 to 20 min with 10–20% B, from 20 to
30 min with 20–30% B, and from 30 to 42 min with 30–42% B. We found that the components of the
chrysanthemum flowers could also be separated with HPLC under weakly basic conditions.
2.2. Retention Rate of 3-CQA and A7G under Basic Conditions
The 3-CQA and A7G were used as targets to investigate the effect of weakly basic conditions on the
retention rate of caffeoylquinic acids and glycosides. Without ammonium hydrogen carbonate, the pH
value of ultrapure water was 6.85 (Figure 2a). At concentrations of 300 and 400 mmol/L NH4HCO3,
the pH value was below 9.0, which indicated that ammonium hydrogen carbonate is weakly basic.

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10
9
a
A
A
B
C
D
8
E
7
6
0
10
100
200
300
400
Concentration (mmol/L)
150
100
50
3-CQA
A7G
b
A A
A
A
A
A
A
B
C
D
E
E
0
0
10
100
200
300
400
Concentration (mmol/L)
3-CQA
A7G
150
100
50
c
A
A
A
A
A A
A
B
C
D
E
F
0
0
0.5
1
1.5
2
2.5
Time (hour)
Figure 2. The effect of basic conditions on the retention rates of 3-CQA and A7G. Results using:
) pH values of ammonium hydrogen carbonate, ( ) various concentrations of ammonium hydrogen
carbonate ranging from 0 to 400 mmol/L, and ( ) time from 0 to 2.5 h. Means indicated by different
(
a
b
c
letters differed significantly with a value of p < 0.01.
The retention rates of 3-CQA decreased as the concentration of the ammonium hydrogen
carbonate increased. Figure 2b shows that the retention rate of 3-CQA decreased to 49.2% when mixed
with 10 mmol/L NH4HCO3 for 1 h. The retention rate of 3-CQA with 10 mmol/L NH4HCO3 was
significantly lower than it was without the treatment with ammonium hydrogen carbonate, p < 0.01.

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The concentrations of ammonium hydrogen carbonate had no significant effect on the retention
rates of A7G, p < 0.01. All of the retention rates for A7G were over 90% when mixed with different
concentrations of ammonium hydrogen carbonate for 1 h. Unlike the acidic conditions, the weakly
basic conditions did not lead to the hydrolysis of glycosides.
The retention rate of 3-CQA was significantly affected by time, p < 0.01. Figure 2c shows that the
retention rates of 3-CQA mixed with 10 mmol/L NH4HCO3 decreased with increasing time. The time
had no significant effect on the retention rates of A7G with 10 mmol/L NH4HCO3, p < 0.01. All the
results showed that the weakly basic conditions could be used for separating flavonoids and glycosides
but they are not be suitable for the purification of caffeoylquinic acids.
2.3. HPLC and UV Analysis
Ammonium acetate aqueous solutions have a pH of 7, so they could be used to eliminate the effect
of acidic and basic conditions on 3-CQA and A7G. The gradient elution solvents consisted of phase A
(water with 10 mmol/L NH4CH3CO2) and phase B (acetonitrile), using the same procedure as described
for the weakly basic conditions. Without pretreatment with ammonium hydrogen carbonate, there
was only one peak for 3-CQA and one peak for A7G in HPLC, as seen in Figure 3. With pretreatment
with ammonium hydrogen carbonate, peak 5 in Figure 3 is consistent with the retention time and
ultraviolet spectrum of A7G, indicating that A7G was stable in weakly basic conditions, but there were
three new peaks generated (peaks 1, 2, and 4 in Figure 3).
mAu
3-CQA
150
100
50
A7G
H2O
2
1
5
3
NH4HCO3
4
0
min
0
10
2
20
30
40
1
4
5
3
nm
300
300
400
300
400
300
400
400
400
300
Figure 3. HPLC and UV analysis of the hydrolysates of 3-CQA and A7G treated with ammonium
hydrogen carbonate. The UV spectrum is from a scan from 210 to 420 nm.
The new peaks 1 and 4 in the UV spectrum were consistent with structure of 3-CQA, while the
retention time was different, indicating that they were the isomers of 3-CQA. The 1-CQA, 4-CQA,
and 5-CQA were the major 3-CQA isomers [13–15]. The retention time and UV spectra of peak 3 was
consistent with 3-CQA, but it is unclear whether this was due to the remaining 3-CQA or an isomer.
Farah [16] studied the effect of roasting on the formation of chlorogenic acid lactones in coffee and
found that roasting causes the isomerisation of chlorogenic acids prior to the formation of lactones,
and that the levels of lactones in roasted coffee do not reflect the levels of precursors in green coffee.
Xie [17] investigated the isomeric transformations of chlorogenic acid in buffers by UPLC-Q-TOF-MS

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and proved that in a phosphate buffer (pH 7.4), trans-5-CQA first isomerises to trans-4-CQA and then
to trans-3-CQA by intramolecular acyl migration. When exposed to UV light, trans-3, -4, and -5-CQA
undergo cis/trans isomerisation to form cis isomers. The isomerisation was dependent on the pH and
the incubation temperature.
2.4. UPLC-Q-TOF-MS Analysis
The hydrolysis behaviour of 3-CQA was further investigated by UPLC-Q-TOF-MS. The formula
and mass of hydrolysates were calculated using the MassHunter Workstation software (2014, version
B.07.00, Agilent Technologies, Inc., Santa Clara, CA, USA). Figure 4 shows that the three hydrolysates
had (1) a formula C8H8O5 and a mass of 183.0294 [M
−
H]−, (2) a formula C5H8O7 and a mass of
H]−. The compound with
179.0196 [M
−
H]−, and (3) a formula C7H12O6 and a mass of 191.0536 [M
−
the formula C5H8O7 had product ion m/z ratios of 161.0076, 99.0076, and 59.0131. The compound with
the formula C7H12O6 had product ion m/z ratios of 85.0290, 93.0340, and 127.0386. The fact that these
results had a highly matching score with the results of a search of the METLIN metabolite database
(supported by Agilent Technologies) led to the conclusion that the formulas C5H8O7 and C7H12O6
represented caffeic acid and quinic acid.
Figure 4. Target MS/MS analysis by UPLC-Q-TOF-MS of the hydrolysates of 3-CQA treated with
ammonium hydrogen carbonate. MS/MS data for 3-CQA was obtained in negative ESI mode. MS/MS
data was for collision energies of 10 eV, 20 eV, and 30 eV. Cpd represents compound. Compounds 1, 4
and 5 were identified as 3,4-dihydroxymandelic acid, caffeic acid, and quinic acid, respectively.

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The compound of formula C8H8O5 had a major ion m/z ratio of 183.0294 [M
ion m/z ratios of 165.0187, 139.0388, 123.0088, and 69.0337. The previous literature [13–17] focused
−
H]− and product
on the isomers of 3-CQA, while there was no literature about this hydrolysate of 3-CQA (peak 1 in
Figure 3). We first used human metabolome databases (HMDB, http://www.hmdb.ca/) and Scifinder
(https://scifinder.cas.org/) to search for this hydrolysate of 3-CQA. A comparison with the MS and MS/MS
data led to the conclusion that this hydrolysate (peak 1 in Figure 3) was 3,4-dihydroxymandelic acid.
Figure 5 shows the hydrolysis pathways of 3-CQA. There were two hydrolysis pathways of
chlorogenic acid in basic conditions, hydrolysation and isomerisation. Besides the isomeric transformations,
the hydrolysates of 3-CQA are caffeic acid, quinic acid, and 3,4-dihydroxymandelic acid.
3-CQA
Hydrolyzation
Isomerization
Quinic acid
1-CQA
Caffeic acid
4-CQA
5-CQA
3,4-dihydroxymandelic acid
Figure 5. The proposed hydrolysation and isomerisation of 3-CQA in weakly basic conditions. 1-CQA
represents 1-caffeoylquinic acid, 3-CQA represents chlorogenic acid, 4-CQA represents 4-caffeoylquinic
acid, and 5-CQA represents 5-caffeoylquinic acid.

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3. Materials and Methods
3.1. Materials
Six kinds of Chrysanthemum morifolium cultivars were purchased from different companies (Table 1).
Apigenin-7-O-glucoside, chlorogenic acid, formic acid, acetonitrile, ammonium hydrogen carbonate,
and ammonium acetate were obtained from Sigma-Aldrich (St Louis, MO, USA). Ultrapure water
(18.2 M
Ω cm) was further purified using a Milli-Q system (Millipore, Billerica, MA, USA). Unless
otherwise mentioned, the chemicals and reagents used in this experiment were of analytical grade.
Table 1. Samples and origins of the studied Chrysanthemum morifolium flower tea from China.
Samples
Codes
GJ
Company
Origins
Beijing Tongrentang Health
Pharmaceutical Industry Co., Ltd.
Beijing Tongrentang Health
C. morifolium ‘Gongju’
C. morifolium ‘Hangbaiju’
C. morifolium ‘Taiju’
C. morifolium ‘Boju’
C. morifolium ‘Chuju’
C. morifolium ‘Huangju’
Huangshan City, Anhui Province
HBJ
TJ
Tongxiang City, Zhejiang Province
Pharmaceutical Industry Co., Ltd.
Zhongwei City, Ningxia Hui
Autonomous Region
Beijing Zhang Yiyuan Jinqiao Tea Co., Ltd.
Bozhou Zhongyitang Chinese Medicinal
Materials Sales Co., Ltd.
Anhui Jutai Chuju Herb Science and
Technology Co., Ltd.
Huangshan Dingxiangwu Ecological
Agriculture Development Co., Ltd.
BJ
Bozhou City, Anhui Province
Chuzhou City, Anhui Province
Shangrao City, Jiangxi Province
CJ
HJ
3.2. Sample Preparation
In this study, the flowers of C. morifolium ‘Gongju’, C. morifolium ‘Hangbaiju’, C. morifolium ‘Tai’,
C. morifolium ‘Boju’, C. morifolium ‘Chuju’ and C. morifolium ‘huangju’ were ground to a powder and
screened through a 100-mesh filter. The samples were stored at −80 ◦C before analysis.
3.3. Extraction with Ultrasound Method
A previously reported ultrasound method [10] was used to extract the components of the
chrysanthemums. Initially, 0.1000
±
0.0005 g of chrysanthemum powder was added to a 2 mL
polyethylene tube, and 1 mL of ultrapure water was added. After being vortexed, the tube was
placed in an ultrasonic cleaner for rapid extraction. The ultrasound conditions were set as follows:
an extraction time of 35 min, a temperature of 50 ◦C, and an ultrasound power of 350 W. Then the
solution was centrifuged with a high-speed refrigerated centrifuge (CR 21G III, Hitachi, Chiyoda-Ku,
Japan) at 10,000 rpm for 1 min. The resulting extracts were filtered through a 0.22
before analysis.
µm nylon membrane
3.4. Separation of the Components under Weakly Basic Conditions by HPLC
Analytical HPLC was performed using an Agilent 1260 HPLC equipped with a degasser,
quad pump, autosampler, and diode array detector (DAD). Samples were scanned (210–420 nm) with
stored wavelengths of 327 nm and 254 nm. In acidic conditions, the components were analysed
according to a previously described method [10] with a Venusil ASB C18 column (250
×
4.6 mm i.d.,
L,
5
µm, Agela Technologies, Tianjin, China) with a flow rate of 1 mL/min, an injection volume of 10 µ
and a temperature of 30 ◦C. The gradient elution solvents composed of phase A (water with 0.1%
formic acid) and phase B (acetonitrile) were as follows: 0–25 min with 20–20% B, 25–30 min with
20–50% B, 30–35 min with 50–100% B, 35–37 min with 100–20% B, and 37–42 min with 20% B.
In weakly basic conditions, the gradient elution solvents consisted of phase A (water with
10 mmol/L ammonium hydrogen carbonate) and phase B (acetonitrile). The analytical conditions were
optimised as follows: 0–10 min with 10–10% B, 10–20 min with 10–20% B, 20–30 min with 20–30%
B, and 30–42 min with 30–42% B. Considering the tolerance of the column, another column (Athena

Molecules 2019, 24, 297
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C18-WP, 250
analyse the components under basic conditions.
×
4.6 mm i.d., 5
µ
m, Anpel Laboratory Technologies Inc., Shanghai, China) was used to
3.5. Hydrolysis Behaviour of 3-CQA and A7G under Basic Conditions.
The hydrolysis behaviour of 3-CQA and A7G were investigated under different conditions
(time and content of ammonium hydrogen carbonate). First, 10
10 L A7G solution (0.25 mg/mL) were mixed with 180 L NH4HCO3 with concentrations ranging
from 10 to 400 mmol/L, and the solutions were kept in a water bath at 50 ◦C for different lengths of time
(0.5 to 2.5 h). A UPLC column (ACQUITY UPLC BEH C18, 2.1 100 mm i.d., 1.7 m, Waters, Dublin,
µL 3-CQA solution (0.125 mg/mL) and
µ
µ
×
µ
Ireland) was used to shorten the analysis time. Because an aqueous ammonium acetate solution is
nearly neutral, it was used to eliminate the possible effects of acidic and basic conditions on 3-CQA
and A7G. The two solvent phases were phase A (water with 10 mmol/L CH3COO NH4) and phase B
(acetonitrile), and the analysis conditions were 0.1 mL/min with an injection volume of 5 µL and an
isocratic elution with phase A and phase B (75:25, v/v). The retention rates of 3-CQA and A7G were
calculated with and without ammonium hydrogen carbonate using the following equation:
Retention rate (%) = A1/A0 × 100
where A1 represents the peak area calculated for mixed with different concentrations of ammonium
hydrogen carbonate, and A0 represents the peak area without the treatment of ammonium hydrogen
carbonate.
3.6. Analysis of the Hydrolysates of 3-CQA and A7G with HPLC and UV
The hydrolysates of 3-CQA and A7G were analysed with HPLC. The two-phase solvents consisted
of phase A (water with 10 mmol/L CH3COONH4) and phase B (acetonitrile), and a column (Athena
C18-WP, 250
×
4.6 mm i.d., 5 µm, Anpel Laboratory Technologies Inc., Shanghai, China) was used
to analyse the hydrolysates. The gradient elution was the same as for the weakly basic conditions,
which were described in Section 3.4. The UV spectrum was determined with a diode array detector
(DAD) that scanned from 210 to 420 nm.
3.7. UPLC-Q-TOF-MS Analysis
The hydrolysates were further analysed with a UPLC-Q-TOF-MS system (1290 Infinity II, G6530C
Q-TOF, Agilent). The UPLC analysis was done with a UPLC column (ACQUITY UPLC BEH C18,
2.1
×
100 mm i.d., 1.7 µm, Waters, Ireland), a phase A of water with 10 mmol/L CH3COO NH4, a phase
B of acetonitrile, and an isocratic elution with phase A and phase B (95:5, v/v). The MS conditions
were a gas temperature of 250 ◦C, a drying gas flow of 7 L/min, a sheath gas temperature of 350 ◦C, a
sheath gas flow of 11 L/min, a capillary voltage of 3500 V, a nozzle voltage of 0 V, a fragmentor voltage
of 130 V, and a skimmer voltage of 65 V. The MS range was 100–1700 m/z with an acquisition rate of
4 spectra/s and an acquisition time of 250 ms/spectrum. The MS/MS range was 100–1000 m/z with
an acquisition rate of 3 spectra/s and an acquisition time of 333.3 ms/spectrum. The targeted MS/MS
fixed collision energies were 10 eV, 20 eV, and 30 eV. The MS data was qualitatively analysed using
MassHunter Workstation software (version B.07.00, coupled with the METLIN metabolite database
supported by Agilent Technologies.
4. Conclusions
Due to the problem of the hydrolysis of glycosides when separating the components of
Chrysanthemum morifolium in acidic conditions, weakly basic conditions were investigated for this
separation. Ammonium hydrogen carbonate aqueous solutions are weakly basic, and because
NH4HCO3 decomposes to form easily-separated NH3, CO2, and H2O, NH4HCO3 solutions were used
as the HPLC solvent. HPLC analysis showed that the components from Chrysanthemum morifolium

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could be separated with weakly basic conditions. As with the purification with acidic solutions,
weakly basic conditions could hydrolyse caffeoylquinic acids, but they have no effect on flavonoids and
glycosides. There were two hydrolysis pathways of chlorogenic acid in basic conditions: hydrolysation
and isomerisation. The 3-CQA could be isomerised to 1-CQA, 4-CQA, and 5-CQA, and caffeic acid,
quinic acid, and 3,4-dihydroxymandelic acid have been proposed as hydrolysates of 3-CQA. The results
showed that weakly basic conditions could be used for the purification of flavonoids and glycosides,
but not for caffeoylquinic acids. This study not only provided a procedure for the separation and
purification of flavonoids and glycosides from Chrysanthemum morifolium in weakly basic conditions,
it also clarified the hydrolysis behaviour of caffeoylquinic acids, which could be helpful for further
investigations into their functional aspects.
Author Contributions: Y.W. (Yuxiao Wang) and Y.N. conceived and designed the experiments; Y.W. (Yuxiao Wang),
Z.X. and Y.W. (Yue Wu) performed the experiments; Y.W. (Yuxiao Wang), M.L., S.P. and Z.L. analysed the data;
Y.W. (Yuxiao Wang) wrote the paper. All authors read and approved the final manuscript.
Funding: This work was supported by the National Natural Science Foundation of China (No. 31871807),
the Special Fund for Agro-Scientific Research in the Public Interest (No. 201503142) and the National Students’
Innovation and Entrepreneurship Training Program (No. 201810019094).
Acknowledgments: The authors appreciate the help of Jianzhong Li (Engineer from Agilent). The authors also
thank the editor Mark Bishop (Innobuddy co., LTD) who checked the language.
Conflicts of Interest: The authors declare no conflict of interest.
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