Panax ginseng - Shimadzu Scientific Instruments

Panax ginseng - Shimadzu Scientific Instruments

Simultaneous Determination
of Major Components in
Panax ginseng using Ultra
Fast Liquid Chromatography
Tadayuki Yamaguchi, Kenichiro Tanaka,
Takeshi Goto, Hirohisa Mikami, Shimadzu Corporation,
380-1, Horiyamashita, Hadano, Kanagawa, Japan
Introduction
The Prominence UFLC has been designed to achieve ultra fast liquid
chromatography by utilizing the Prominence HPLC series. At this point, we have
successfully demonstrated high speed and separation performance without
resorting to high pressure.
Panax ginseng is one of the most useful Chinese herbal medicines due to its
various biological or pharmacological activities, such as anti-aging and antioxidation. Among its active constituents, ginsenosides are an important category
of bioactive compounds that may possess anticarcinogenic properties, as well as
tranquilizing effects on the central nervous system.
Accordingly, the determination of ginsenosides in Panax ginseng for efficacy and
safety evaluations has gained much attention within pharmaceutical and food
sciences.
The separation and detection methods for ginsenosides typically involve
conventional HPLC analysis using 250 mm columns packed with 5 μm particles.
To achieve an efficient and rapid analysis, however, would require various
methodological considerations to optimize the analytical conditions.
In this paper, we present a high-speed and high-resolution HPLC method for
the simultaneous determination of five major ginsenosides (ginsenoside Rg1,
Re, Rb1, Rc, and Rd) in Panax ginseng using shorter columns packed with
smaller-sized particles. Prior to the HPLC analysis, solid phase extraction (SPE)
was performed for the methanol extracts of Panax ginseng powder in order to
remove the complex contaminants.
Experimental
1) Sample
Panax ginseng ( White ginseng ) was purchased from Yamamoto Kanpou
Seiyaku Co.,Ltd (Tokyo, Japan). Ginsenosides standards (ginsenoside
Rg1, Re, Rb1, Rc, and Rd) were purchased from Wako Pure Chemical
Industries (Osaka, Japan) .
2) Instruments
The Prominence UFLC system consisted of two LC-20AD pumps,
DGU-20A5 degasser, SIL-20AC autosampler, CTO-20AC column oven,
SPD-20AV UV-Visible detector, and LCMS-2010EV mass spectrometer
(Shimadzu Corporation, Kyoto, Japan).
3) HPLC column and SPE cartridge
“Luna C18”, “Synergi Polar-RP”, and “strata-X ” were purchased from
Phenomenex Inc. (Torrance, CA, USA).
Effects of Smaller Particle Sizes on Chromatography
20
45
5 μm
3 μm
Sub 2 μm
16
40
35
Column Pressure (MPa)
18
14
30
HETP
12
25
10
20
8
15
6
4
10
2
5
0
0
0
5
10
15
20
25
30
35
40
45
Interstitial Linear Velocity (cm/min)
Fig. 1: van Deemter plots with different
particle size packing material columns
50
0
1
2
3
4
5
Particle Size (μm)
Fig. 2: Relationship between particle size and
column pressure
6
Structures of Ginsenosides
R3
OH
R1
Compound
Ginsenoside Rg1
Ginsenoside Re
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
R1
OH
OH
O-Glc(2-1)Glc
O-Glc(2-1)Glc
O-Glc(2-1)Glc
R2
R2
O-Glc
O-Glc(2-1)Rha
H
H
H
R3
O-Glc
O-Glc
O-Glc(6-1)Glc
O-Glc(6-1)Araf
O-Glc
Glc:
Rha:
Araf:
Exact Mass
800.49
946.55
1108.60
1078.59
946.55
β-D-glucose
α-L-rhamnose
α-L-arabinose (furanose)
Analysis of Ginsenosides using a C18 Column
mAU
30
■Peaks
1. Ginsenoside Rg1
2. Ginsenoside Re
3. Ginsenoside Rb1
4. Ginsenoside Rc
5. Ginsenoside Rd
Luna 2.5μm C18(2)-HST (50 mmL.×2.0 mmI.D., 2.5μm)
1+2
20
3
4
5
10
0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
min
Fig. 3: Chromatogram of a standard mixture of 5 ginsenosides with a C18 column (50 mg/L each)
Chromatographic conditions
Column
: Phenomenex Luna 2.5μm C18(2)-HST (50 mm L x 2.0 mmI.D.,2.5 μm)
Flow rate
: 0.6 mL/min
Column temp.
: 35Ԩ
Mobile phase
: A) Water
B) Acetonitrile
Time prog.
: B. Conc 10% (0 min) → 40% (10 min)
Injection vol.
: 2 μL
Detection
: SPD-20AV (203 nm)
Analysis of Ginsenosides with Polar-RP
15.0
mAU
■Peaks
1. Ginsenoside Rg1
2. Ginsenoside Re
3. Ginsenoside Rb1
4. Ginsenoside Rc
5. Ginsenoside Rd
Synergi Polar-RP 80Å (250 mm L.×4.6 mmI.D., 4 μm)
1
10.0
2
5
3 4
5.0
0.0
0
mAU
15.0
10
20
30
40
50
60
70
min
Synergi 2.5μm Polar-RP 100Å (50 mm L.×2.0 mmI.D., 2.5 μm)
1
10.0
3 4
2
5
5.0
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Mobile phase:
A) Water B) Acetonitrile
Flow rate:
Upper) 1.5 mL/min ,
Lower) 0.6 mL/min
Column temp : 35Ԩ
Time prog:
Upper) :B. Conc 15%(0 min) → 20%(30 min)
→25%(40 min) → 30%(80 min)
Lower) :B. Conc 15%(0 min) → 20%(3 min)
→25%(4 min) → 30%(8 min)
Injection vol: Upper) 10 μL, Lower) 2 μL
Detection: SPD-20AV ( 203 nm )
Flow cell: Upper) Conventional cell
Lower) Semi-micro cell
Fig. 4: Chromatograms of a standard
mixture of 5 ginsenosides (50 mg/L
each); (Upper: Synergi Polar-RP 80Å,
min Lower: Synergi 2.5 μm Polar-RP 100Å)
Benefits of Polar-Embedded and/or Polar Endcapping
Column
Stationary phase
Luna 2.5µm C18(2)-HST
ODS
Synergi 2.5µm Polar-RP
Ether-linked phenyl
with polar endcapped
Particle size
Pore size Surface area Carbon Loading
(µm)
2.5
(nm)
10
(m /g)
400
2
(%)
17.5
2.5
8
475
11
“Synergi Polar-RP” is an ether-linked phenyl phase with proprietary hydrophilic
endcapping designed specifically to maximize retention and selectivity for polar
and aromatic analytes.
Retention mechanisms include hydrogen bonding, dipole-dipole, and
aromatic (π-π) interactions. These features allow for improved polar retention
that complements conventional C18 column chemistries, as well as provide
improved peak shape and alternative selectivity compared to other polar
phases.
Calibration Curves for Ginsenosides
700000
Ginsenoside Rg1
Ginsenoside Re
Ginsenoside Rb1
600000
Peak Area
500000
Ginsenoside Rc
Ginsenoside Rd
400000
300000
200000
100000
0
Compound
Ginsenoside Rg1
Ginsenoside Re
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
0
250
500
750
Concentration (mg/L)
Linear Range
(mg/L)
10　-　1000
10　-　1000
10　-　1000
10　-　1000
10　-　1000
r
2
0.9996
0.9996
0.9996
0.9996
0.9996
1000
LOQ
(ng)
4.6
6.7
7.1
7.4
7.0
Fig. 5: Calibration curve
(UV detection)
LOD
(ng)
1.5
2.2
2.4
2.4
2.3
Table 1: Linearity,
LOQ, and LOD for
ginsenosides
(2 μL inj.)
Repeatability (UV Detection)
Table 2: Repeatability for ginsenosides
Compound
1st
2nd
3rd
4th
5th
6th
Average
%RSD
Ginsenoside Rg1
Ginsenoside Re
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
R.T.
(min)
R.T.
(min)
R.T.
(min)
R.T.
(min)
R.T.
(min)
3.33
3.33
3.32
3.32
3.32
3.32
3.32
0.08
Area
(uV･ sec)
7497
7491
7585
7445
7443
7452
7486
0.72
3.59
3.60
3.59
3.59
3.59
3.59
3.59
0.08
Area
(uV･ sec)
5172
5238
5204
5200
5108
5126
5175
0.96
6.49
6.49
6.49
6.49
6.48
6.49
6.49
0.03
Area
(uV･ sec)
4945
4909
4948
5009
4940
5008
4960
0.81
6.83
6.83
6.84
6.84
6.83
6.83
6.83
0.04
Area
(uV･ sec)
4886
4905
4923
4910
4944
4950
4920
0.50
7.82
7.82
7.83
7.83
7.82
7.82
7.82
0.05
Area
(uV･sec)
5875
5919
5865
5881
5828
5933
5884
0.65
(10 mg/L, 2 μL inj.)
High repeatability at extremely low concentrations
Official Sample Preparation (JP* Method)
Sample 1.0 g
Dissolve in 60% Methanol (aq.) (30 mL)
Sonicate (15 min)
Centrifuge (3000 rpm, 5 min)
Supernatant 1
Residue 1
Dissolve in 60 % methanol (aq.) (15 mL)
Sonicate (15 min)
Centrifuge (3000 rpm, 5 min)
Supernatant 2
Residue 2
Supernatants 1+2
Adjust to 50 mL using 60% methanol (aq.)
Waste
HPLC or LC/MS
*Japanese Pharmacopoeia
HPLC Results (JP Method Sample Preparation only)
mAU
■Peaks
1. Ginsenoside Rg1
2. Ginsenoside Re
3. Ginsenoside Rb1
4. Ginsenoside Rc
5. Ginsenoside Rd
20.0
1
15.0
10.0
Table 3: Concentration of
ginsenosides in Panax ginseng
3
Compound
ginsenoside Rg1
2.62
ginsenoside Re
1.08
ginsenoside Rb1
3.18
ginsenoside Rc
0.96
ginsenoside Rd
0.23
2
Total
5.0
4
5
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
Content
(mg/g)
8.0 min
Fig. 5: Chromatogram of Panax ginseng after sample preparation (JP)
8.07
Additional Sample Preparation (SPE Cleanup)
Sample Preparation (JP Method)
2 mL Aliquot
strata-X
(60 mg/3 mL)
Compared to standard C18 sorbents,
the strata-X reversed phase sorbent retains
analytes by a combination of hydrophobic,
hydrogen-bonding, and π-π interations for
enhanced retention of polar compounds.
Dilute with water to 10 mL
Condition with methanol (2 mL)
Equilibrate with water (2 mL)
Wash with 10% methanol (aq.) (2 mL)
Elute with methanol (2 mL)
HPLC or LC/MS
Result (Additional Sample Preparation)
mAU
Table 4: Concentration of
ginsenosides in Panax ginseng
■Peaks
1. Ginsenoside Rg1
2. Ginsenoside Re
3. Ginsenoside Rb1
4. Ginsenoside Rc
5. Ginsenoside Rd
20.0
15.0
1
10.0
Compound
3
ginsenoside Rg1
2.38
ginsenoside Re
0.88
ginsenoside Rb1
3.25
ginsenoside Rc
0.90
ginsenoside Rd
0.21
Total
4
5.0
2
5
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0 min
Fig. 6: Chromatogram of Panax ginseng after SPE cleanup
Content
(mg/g)
7.62
Effect of Pretreatment with SPE
mAU
Red : Sample without SPE
Blue : Sample with SPE
20.0
1
15.0
■Peaks
1. Ginsenoside Rg1
2. Ginsenoside Re
3. Ginsenoside Rb1
4. Ginsenoside Rc
5. Ginsenoside Rd
10.0
3
2
5.0
4
5
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Fig. 7: Comparative chromatograms showing effect of SPE cleanup
Removal of polar matrix impurities
Improved quantity precision
min
Recovery of Ginsenosides
Table 5: Recovery of ginsenosides
Compound
Ginsenoside Rg1
Ginsenoside Re
Ginsenoside Rb1
Ginsenoside Rc
Ginsenoside Rd
Recovery
(%)
97　-　99
97　-　98
94　-　96
96　-　97
94　-　95
%RSD
0.81
0.55
1.04
0.67
0.51
(n=3, 50 mg/L, 2 μL inj.)
Recovery tests were carried out using the standard mixture of ginsenosides (50 mg/L).
The recovery of ginsenosides after SPE cleanup was nearly quantitative.
LC/MS Analytical Conditions
Instruments
Column
Dimension
Mobile phase
:
:
:
:
Flow rate
Injection vol.
Column temp.
Ionization mode
Applied voltage
Nebulizer gas flow
Drying gas press.
CDL temp.
Heat block temp.
Scan range
Interval
:
:
:
:
:
:
:
:
:
:
:
Selected monitering ion
:
Shimadzu LCMS-2010EV
Phenomenex Synergi 2.5µm Polar-RP 100Å
50 mm L. × 2.0 mm I.D.
A) 5 mmol/L Ammonium acetate aq. B) Acetonitrile
B.Conc 15% (0 min) → 20% (3 min) → 25% (4 min)→ 30% (8 min)
0.6 mL/min
2 μL
35 ℃
ESI negative
-3.5 kV
1.5 L/min
0.1 MPa
250℃
200℃
m/z 600-1250
1 sec / Scan
m/z 859 [M+CH3COO] for Ginsenoside Rg1
m/z 946 [M-H]-
for Ginsenoside Re, Ginsenoside Rd
-
for Ginsenoside Rb1
-
for Ginsenoside Rc
m/z 1108 [M-H]
m/z 1078 [M-H]
MS Spectra of Ginsenosides
Inten.
(x100,000)
Inten.
1: ginsenoside Rg1859.3 [M+CH3
1.00
COO]-
1078.3 [M-H]-
4: ginsenoside Rc
4.0
0.75
(x10,000)
3.0
[M-H]-
0.50
2.0
799.3
0.25
1.0
0.0
0.00
600
700
Inten.
7.5
800
900
1000
1100
1200
600
m/z
(x10,000)
700
Inten.
2: ginsenoside Re
945.5 [M-H]-
800
1000
1100
1200
m/z
(x100,000)
5: ginsenoside Rd
1.25
900
945.5 [M-H]-
1.00
5.0
0.75
1005.3
0.50
2.5
0.25
0.00
0.0
600
700
Inten.
800
900
1000
1100
1200
m/z
600
700
800
900
1000
1100
1200
(x10,000)
613.5
7.0
3: ginsenoside Rb1
6.0
1107.5 [M-H]-
5.0
4.0
3.0
2.0
1.0
0.0
600
700
800
900
1000
1100
1200
m/z
Fig. 8: Mass spectra of ginsenosides
m/z
SIM Chromatogram of Panax Ginseng
(x10,000)
2.0
1
859.00 (1.00)
946.00 (1.00)
1108.00 (1.00)
1078.00 (1.00)
■Peaks
1. Ginsenoside Rg1
2. Ginsenoside Re
3. Ginsenoside Rb1
4. Ginsenoside Rc
5. Ginsenoside Rd
1.5
2
1.0
3
5
0.5
4
0.0
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
Fig. 9: SIM chromatograms of Panax ginseng
Conclusion
¾The UFLC instrumentation was utilized for the simultaneous determination of five major
ginsenosides (Rg1, Re, Rb1, Rc, and Rd) using two reversed phased columns with
different selectivities.
¾Baseline separation was achieved using a Phenomenex Synergi Polar-RP 100Å
column packed with 2.5 μm particles featuring polar-endcapped and polar-embedded
stationary phase, which allows hydrogen-bonding and dipole-dipole interactions in
addition to the main hydrophobic interactions.
¾The ultra fast liquid chromatography method was optimized to achieve an analysis time
of less than 10 minutes (including equilibration), which is about one tenth that of
conventional HPLC analyses.
¾The official sample preparation method was modified by including SPE cleanup step.
Interferences from the Panax ginseng sample matrix were effectively removed, thus
improving the baseline and precision of the analysis.
¾Further optimizations of the UFLC/MS analysis are currently underway.