Page 1
Supporting Information
Observation and Confirmation of Oxidation Reaction Occurring on
Ultra-high Performance Liquid Chromatography Column
Caiming Tang
1,2,*,
Jianhua Tan
3,4,*,
Jiabin Jin 1,2, Shaofeng Xi 4, Huiyong Li 4, Qilai Xie 3,
Xianzhi Peng 1
1
State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese
Academy of Sciences, Guangzhou 510640, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
3
College of Natural Resources and Environment, South China Agricultural University, Guangzhou
510642, China
4
Guangzhou Quality Supervision and Testing Institute, Guangzhou 510110, China
*Corresponding Author.
Caiming Tang
Tel: +86-020-85291489; Fax: +86-020-85290009. E-mail: CaimingTang@gig.ac.cn.
Jianhua Tan
Tel: +86-020-020-83300529; Fax: +86-020-020-83300529. E-mail: tanjianhua0734@aliyun.com.
Page 2
Figure Captions.
Figure S-1. Workflow of the finding, confirmation and explanation of the redox
reaction on UPLC column. Notes: [M-G-H]+ is the product ion of the precursor ion [MH]+ losing a glucoside (G) group, and [M-G-3H]- is the product ion of the precursor ion
[M-3H]- losing a G group; VC: ascorbic acid.
Figure S-2. High resolution mass spectra of baicalin and its oxidized product caused
by on-column oxidation reaction. The precursor ions of baicalein and baicalein oxide
were generated during collision induced dissociation (CID). The detection was
conducted with ESI-Q-TOF HRMS operated in negative mode.
Figure S-3. High resolution mass spectra of baicalin and its oxidized product caused
by oxidation reaction on HPLC columns. Column A was Ultimate® AQ-C18 column
(4.6×150 mm, 3 µm, Welch materials, Shanghai, China), and column B was Waters
XBridgeTM C8 column (4.6×250 mm, 5 µm, Milford, MA, USA). The detection was
conducted with ESI-Q-TOF HRMS operated in negative mode.
Figure S-4. Extracted diode array detector (DAD) spectra at the retention times of
baicalin, baicalein and their on-column oxidized with the wavelength range of 210-400
nm.
Figure S-5. Chromatograms of baicalin and its on-column oxidized product baicalein
oxide on the UPLC column without inlet frit (left panel) and the column without both
the inlet and outlet frits (right panel). The used UPLC column was Waters BEH C18
column (2.1×50 mm, 1.7 µm).
Figure S-6. The ratio of the signal intensity of the oxidized product (baicalin oxide/
baicalein oxide) to the summational signal intensity of corresponding oxidized product
and unchanged parent analyte (baicalin/baicalein) with different elution flow rates. The
used UPLC column was Waters BEH C18 column (2.1×50 mm, 1.7 µm). The ions m/z
269, m/z 271, m/z 445 and m/z 447 correspond to baicalein oxide, baicalein, baicalin
oxide and baicalin, respectively. The blue bars link to the precursor ions of baicalein
and baicalein oxide which were generated during ESI process by baicalin and baicalin
oxide, respectively. The green bars correspond to the intact parent analyte baicalein and
the oxidized product baicalein oxide generated on the UPLC column.
Figure S-7. Chromatograms of baicalin, baicalein and their on-column oxidized
products on the UPLC column used in reversed flow direction. The used UPLC column
was Waters BEH C18 column (2.1×50 mm, 1.7 µm).
Figure S-8. The ratio of the signal intensity of baicalin oxide (m/z 445) to the
summational signal intensity of baicalin oxide and baicalin (m/z 447) with and without
Page 3
the post-column infusion of reducing agent solution ((NH4)2S). The concentration of
the post-column infused (NH4)2S solution was 1.7 mM. The solution was prepared with
ultra-pure water. The flow rates of the post-column infusion were set from 5 μL/min to
50 μL/min.
Figure S-9. The ratio of the signal intensity of baicalein oxide (m/z 269) to the
summational signal intensity of baicalein oxide and baicalein (m/z 271) with and
without the post-column infusion of reducing agent solution ((NH4)2S). The
concentration of the post-column infused (NH4)2S solution was 1.7 mM. The solution
was prepared with ultra-pure water. The flow rates of the post-column infusion were set
from 5 μL/min to 50 μL/min.
Figure S-10. The ratio of the signal intensity of baicalin oxide (m/z 445) to the
summational signal intensity of baicalin oxide and baicalin (m/z 447) with and without
the post-column infusion of reducing agent solutions tested in this study. The reducing
agent solutions contained the respective agent with the concentration of 10 mM,
excepted for (NH4)2S, of which the concentration was 1.7 mM. These solutions were
prepared with ultra-pure water. The post-column infusion flow rates of the reducing
agents were transformed into nmol/min based on the calculation with the concentrations
and flow rates of the post-column infusion.
Figure S-11. The ratio of the signal intensity of baicalein oxide (m/z 269) to the
summational signal intensity of baicalein oxide and baicalein (m/z 271) with and
without the post-column infusion of reducing agent solutions tested in this study. The
reducing agent solutions contained the respective agent with the concentration of 10
mM, excepted for (NH4)2S, of which the concentration was 1.7 mM. These solutions
were prepared with ultra-pure water. The post-column infusion flow rates of the
reducing agents were transformed into nmol/min based on the calculation with the
concentrations and flow rates of the post-column infusion.
Figure S-12. General illustration of the main findings of this study: oxidation reaction
on UPLC column and reduction reaction on ESI source.
Page 4
Table captions
Table S-1. Chemical information of the investigated model compounds.
Table S-2. Gradient elution program I.
Table S-3. Gradient elution program II.
Table S-4. Mass spectrometry working conditions.
Table S-5. Exact mass spectrometry data of the investigated model compounds and
their degradation products caused by on-column oxidation reaction.
Page 5
Tables
Table S-1. Chemical information of the investigated model compounds.
Name
Baicalin
Molecular
Exact molecular
formula
weight (u)
C21H18O11
446.0849
CAS No
Chemical structure
21967-41-9
OH
O
HO
Baicalein
C15H10O5
270.0528
491-67-8
HO
O
HO
Propyl gallate
(PG)
O
C10H12O5
212.0685
121-79-9
HO
O
HO
Rutin
C27H30O16
610.1534
153-18-4
C21H20O11
448.1006
522-12-3
C27H32O14
580.1792
10236-47-2
C20H22O9
406.1264
82373-94-2
quercetin-3rhamnoside
(QR)
Naringin
2,3,5,4’Tetrahydroxy
stilbene-2-Οβ-D-glucoside
(THS-G)
Page 6
Table S-2. Gradient elution program I.
Step
Total time (min)
Flow rate (µL/min)
MPA (%)
MPB (%)
1
Initial
400
70
30
2
1.5
400
30
70
3
1.6
400
70
30
4
2.5
400
70
30
Table S-3. Gradient elution program II.
Step
Total time (min)
Flow rate (µL/min)
MPA (%)
MPB (%)
0
Initial
400
70
30
1
3.5
400
30
70
2
3.6
400
70
30
3
5
400
70
30
Table S-4. Mass spectrometry working conditions.
Parameter
Value
Trap Collision Energy
6
Transfer Collision Energy
4
Collision adduced dissociation
Medium
Capillary Voltage
2500 V
Cone voltage
35 V
Desolvation temperature
400 oC
Ion source temperature
100 oC
Cone Gas 1
50 L/h
Desolvation gas
600 L/h
Mass scan range
100-1000 u
Acquisition mode
centroid
Scan time
0.2 s
Lock Mass
554.2615 u
Acquisition mode
V mode
Page 7
Table S-5. Exact mass spectrometry data of the investigated model compounds and their degradation products caused by on-column oxidation
reaction.
Name
Precursor ion (In ESI+, [M+H]+)
Chemical formula
Theoretical
m/z (u)
[1]
Precursor ion (In ESI-, [M-H]-)
Detected
Mass accuracy
Chemical
Theoretical
m/z (u)
(ppm)
formula
m/z (u)
[1]
Detected
Mass accuracy
m/z (u)
(ppm)
Baicalin
C21H19O11
447.0927
447.0960
7.4
C21H17O11
445.0776
445.0789
4.0
Baicalin oxide
C21H17O11
445.0771
445.0799
6.3
C21H15O11
443.0614
443.0562
-11.7
Baicalein
C15H11O5
271.0606
271.0632
9.6
C15H9O5
269.0455
269.0450
-1.9
Baicalein oxide
C15H9O5
269.0451
269.0451
0
C15H7O5
267.0294
267.0301
2.6
PG
NA
NA
NA
NA
C10H11O5
211.0607
211.0616
4.3
PG oxide
NA
NA
NA
NA
C10H9O5
209.0450
209.0472
10.5
Rutin
NA
NA
NA
NA
C27H29O16
609.1456
609.1451
-0.8
Rutin oxide
NA
NA
NA
NA
C27H27O16
607.1299
607.1323
4.0
QR
NA
NA
NA
NA
C21H19O11
447.0927
447.0915
-2.7
QR oxide
NA
NA
NA
NA
C21H17O11
445.0771
445.0766
-1.1
Naringin
NA
NA
NA
NA
C27H31O14
579.1714
579.1691
-4.0
THS-G
NA
NA
NA
NA
C20H21O9
405.1186
405.1165
-5.2
[1] Patiny, L., Borel, A. ChemCalc: A building block for tomorrow’s chemical infrastructure. Journal of chemical information and modeling, 2013, 53(5), 1223-1228.
Page 8
Figures
Figure S-1. Workflow of the finding, confirmation and explanation of the redox
reaction on UPLC column. Notes: [M-G-H]+ is the product ion of the precursor ion [MH]+ losing a glucuronide (G) group, and [M-G-3H]- is the product ion of the precursor
ion [M-3H]- losing a G group; VC: ascorbic acid.
Page 9
Figure S-2. High resolution mass spectra of baicalin and its oxidized product caused
by on-column oxidation reaction. The precursor ions of baicalein and baicalein oxide
were generated during collision induced dissociation (CID). The detection was
conducted with ESI-Q-TOF HRMS operated in negative mode.
Page 10
Figure S-3. High resolution mass spectra of baicalin and its oxidized product caused
by oxidation reaction on HPLC columns. Column A was Ultimate® AQ-C18 column
(4.6×150 mm, 3 µm, Welch materials, Shanghai, China), and column B was Waters
XBridgeTM C8 column (4.6×250 mm, 5 µm, Milford, MA, USA). The detection was
conducted with ESI-Q-TOF HRMS operated in negative mode.
Page 11
Figure S-4. Extracted diode array detector (DAD) spectra at the retention times of
baicalin, baicalein and their on-column oxidized products with the wavelength range of
210-400 nm.
Page 12
Figure S-5. Chromatograms of baicalin and its on-column oxidized product baicalein
oxide on the UPLC column without inlet frit (left panel) and the column without both
the inlet and outlet frits (right panel). The used UPLC column was Waters BEH C18
column (2.1×50 mm, 1.7 µm).
Page 13
Signal intensity of oxidized product/(signal intensity of oxidized product+signal intensity of intact analyte)
1
Ratio 269/(269+271)
Ratio 445/(445+447)
Ratio 269/(269+271) (Baicalein)
0.9
0.8
0.7
Ratio
0.6
0.5
0.4
0.3
0.2
0.1
0
50 μL/min
200 μL/min
300 μL/min
400 μL/min
600 μL/min
Flow rate
Figure S-6. The ratio of the signal intensity of the oxidized product (baicalin oxide/baicalein oxide) to the summational signal intensity of
corresponding oxidized product and unchanged parent analyte (baicalin/baicalein) with different elution flow rates. The used UPLC column was
Waters BEH C18 column (2.1×50 mm, 1.7 µm). The ions m/z 269, m/z 271, m/z 445 and m/z 447 correspond to baicalein oxide, baicalein, baicalin
oxide and baicalin, respectively. The blue bars link to the precursor ions of baicalein and baicalein oxide which were generated during ESI process
by baicalin and baicalin oxide, respectively. The green bars correspond to the intact parent analyte baicalein and the oxidized product baicalein
oxide generated on the UPLC column.
Page 14
Figure S-7. Chromatograms of baicalin, baicalein and their on-column oxidized
products on the UPLC column used in reversed flow direction. The used UPLC column
was Waters BEH C18 column (2.1×50 mm, 1.7 µm).
Page 15
Signal intensity of m/z 445/(signal intensity of m/z 445+signal
intensity of m/z 447)
0.7
0.6
Ratio
0.5
0.4
0.3
0.2
0.1
0
Non-infusion
5 μL/min
10 μL/min
20 μL/min
30 μL/min
40 μL/min
50 μL/min
Flow rate of post-column infusion of (NH4)2S solution (1.7 mM in pure
water)
Figure S-8. The ratio of the signal intensity of baicalin oxide (m/z 445) to the
summational signal intensity of baicalin oxide and baicalin (m/z 447) with and without
the post-column infusion of reducing agent solution ((NH4)2S). The concentration of
the post-column infused (NH4)2S solution was 1.7 mM. The solution was prepared with
ultra-pure water. The flow rates of the post-column infusion were set from 5 μL/min to
50 μL/min.
Page 16
Signal intensity of m/z 269/(signal intensity of m/z 269+signal
intensity of m/z 271)
1
0.9
0.8
0.7
Ratio
0.6
0.5
0.4
0.3
0.2
0.1
0
Non-infusion
5 μL/min
10 μL/min
20 μL/min
30 μL/min
40 μL/min
50 μL/min
Flow rate of post-column infusion of (NH4)2S solution (1.7 mM in pure water)
Figure S-9. The ratio of the signal intensity of baicalein oxide (m/z 269) to the
summational signal intensity of baicalein oxide and baicalein (m/z 271) with and
without the post-column infusion of reducing agent solution ((NH4)2S). The
concentration of the post-column infused (NH4)2S solution was 1.7 mM. The solution
was prepared with ultra-pure water. The flow rates of the post-column infusion were set
from 5 μL/min to 50 μL/min.
Page 17
Signal intensity of m/z 445/(signal intensity of m/z 445+signal
intensity of m/z 447)
0.9
0.8
0.7
Ratio
0.6
0.5
0.4
0.3
0.2
0.1
0
Flow rate of post-column infusion of reducing agent solutions (disolved in
pure water)
Figure S-10. The ratio of the signal intensity of baicalin oxide (m/z 445) to the
summational signal intensity of baicalin oxide and baicalin (m/z 447) with and without
the post-column infusion of reducing agent solutions tested in this study. The reducing
agent solutions contained the respective agent with the concentration of 10 mM,
excepted for (NH4)2S, of which the concentration was 1.7 mM. These solutions were
prepared with ultra-pure water. The post-column infusion flow rates of the reducing
agents were transformed into nmol/min based on the calculation with the concentrations
and flow rates of the post-column infusion.
Page 18
Signal intensity of m/z 269/(signal intensity of m/z 269+ signal
intensity of m/z 271)
1.2
1
Ratio
0.8
0.6
0.4
0.2
0
Flow rate of post-column infusion of reducing agent solutions (disolved in
pure water)
Figure S-11. The ratio of the signal intensity of baicalein oxide (m/z 269) to the
summational signal intensity of baicalein oxide and baicalein (m/z 271) with and
without the post-column infusion of reducing agent solutions tested in this study. The
reducing agent solutions contained the respective agent with the concentration of 10
mM, excepted for (NH4)2S, of which the concentration was 1.7 mM. These solutions
were prepared with ultra-pure water. The post-column infusion flow rates of the
reducing agents were transformed into nmol/min based on the calculation with the
concentrations and flow rates of the post-column infusion.
Page 19
Figure S-12. General illustration of the main findings of this study: oxidation reaction
on UPLC column and reduction reaction on ESI source.
Oxidation reactions of polyphenolic compounds can occur on UPLC columns, which
could pose negative impact to the quantitative and qualitative analysis of these
compounds. Fortunately, the oxidation products can be reduced back to their parent
forms during the ESI process with the presence of reducing agents.