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3. Lomonosov Moscow State University Analytical chemistry division
Chemistry Department
Analytical chemistry division
THE USE OF LINEAR ION TRAP FOR RAPID ONE-RUN GINSENOSIDE PROFILING IN ROOTS AND GINSENG BASED PRODUCTS
Dr. Igor Rodin
(Andrey Stavrianidi; Elena Stekolshchikova; Ivan Godovikov; Oleg Shpigun)
Beijing

4. Ginsenosides 1 Sapogenin: Sugar moieties: Substituents: ППТ: 476H
ППТ: 476
ППД: 460
ОАК: 456
Sugar moieties:
Substituents:

5. Isomeric ginsenosides2
R & S ginsenosides (20(S)Rh1 и 20(R)Rh1)
Double bond position isomers (F4 и Rg6)
Ara (p), Ara (f), Xyl – isomers (Rb2, Rb3, Rc и Ra1, Ra2)
Sugar chain position isomers (Rh1 и F1)
Sugar moieties position isomers (Rg1 и Rf)
Isomeric pseudoginsenosides and ginsenosides (Rf и F11)
PPT-Rha / PPD-Glc isomers (Re и Rd)
Side chain C17 and sapogenin isomers

6. Ginsenoside trasformation3
Ginsenoside trasformation
Steaming
PPT
t °C
(Chang Y.H. et al., 2009) Re
Rh1
PPD
t °C
(Wang C.Z. et al., 2006)
Rg3
Rh2

7. Ginsenoside trasformation4
Ginsenoside trasformation
t °C
Soxhlet
(Du X.W. et al., 2004)
- H2O
(Wang C.Z. et al., 2007)
Rg3
Rk1
437 / 623
W.-Z. Yang et al. Analytica Chimica Acta 739 (2012) 56

8. Ginsenoside transformation4
Ginsenoside transformation

9. 5
Goal of the study
Development of new detection and LC-MS/MS identification methods for bioactive ingredients analysis in the ginseng based on fragmentation pathways and other features of the mass spectrometric behavior of the ginsenosides.
Evaluation of the analytical capabilities of the approaches developed.

10. 6
Objectives
Ionization and fragmentation process study of the ginsenosides using HPLC-MS/MS system. Optimization of the conditions for obtaining the mass spectra that contain information about the structure of the compound.
Algorithm development for identification of structural fragments as well as the evaluation of significant features that can be used to classify unknown components in the study of ginseng saponins with different types of sapogenin.
Selection and optimization of sample preparation conditions for the effective and non-destructive extraction of the ginsenosides from plant materials and ginseng based products.
Study of the chromatographic behavior of the ginsenosides in a reverse- phase HPLC.

11. Ionization and fragmentation process study of the ginsenosides7

12. Ginsenoside mass spectra8
Ginsenoside mass spectra
[M-H]-
Rb1, ESI(-), Q1 scan PPD-20-Glc-Glc-3-Glc-Glc
[M+2H2O-H]-
Intensity, cps.
[M-Glc-H]-
[M-GlcGlc-H]-
m/z, Da

13. Ginsenoside mass spectra9
Ginsenoside mass spectra
[M-H]-
Rb1, ESI(-), MS/MS (CE -50 V) PPD-20-Glc-Glc-3-Glc-Glc
Intensity, cps.
[M-Glc-H]-
[M-GlcGlcGlc-H]-
[M-GlcGlc-H]-
m/z, Da

14. Pseudoginsenoside mass spectra10
Pseudoginsenoside mass spectra
RT5, ESI(-), MS/MS (CE -50 V) OT-6-Glc
[M-H]-
Intensity, cps.
m/z, Da

15. Ginsenoside mass spectra11
Rс, ESI(+), Q1 scan
PPD-20-GlcAra(f)-3-GlcGlc
Rg1, ESI(+), Q1 scan
PPT-20-Glc-6-Glc
(100%)
409 (18%)
[M+Na]+
441 (12%), 443 (14 %)
m/z, Да
[M+Na]+
>1 Da
m/z, Да
Mass spectra registration in the ESI-LITMS scan mode (LIT – Linear Ion Trap)

16. Ginsenoside mass spectra12
Ginsenoside mass spectra
Rg1, ESI(+), LIT scan
Rg1, ESI(+), Q1 scan
[M+Na]+
<0.5 Da
[M+Na]+
m/z, Да
Rg1, APCI(+), LIT scan
m/z, Да
CE?
DP = 70 V, EP = 10 V, CE = 5 V
m/z, Да

17. Algorithm development for identification of structural fragments and ginsenoside fragmentation pattern study 13

18. Ginsenoside mass spectra features14
Ginsenoside mass spectra features 3
Glc+H2O 4
Intensity, cps.
Rg1 2
M-Glc-H2O+H
1 - M-GlcGlc-H2O+H
2 - M-GlcGlc-2H2O+H
3 - M-GlcGlc-3H2O+H
4 - M-GlcGlc-4H2O+H
M-Glc-2H2O+H
M+Na 1
M-Glc-3H2O+H
M+K
m/z, Da

19. Substituent position isomers15
Substituent position isomers
Rh1 F1
M-H2O+H
M-2H2O+H
M-H2O+H
m/z, Da
m/z, Da

20. Sugar moieties position isomers16
Sugar moieties position isomers
Intensity, cps.
Rg1
m/z, Da
Intensity, cps. Rf
M-2H2O+H
m/z, Da

21. PPT-Rha / PPD-Glc (Re и Rd)17
PPT-Rha / PPD-Glc (Re и Rd)
Mr (Re) = Mr (Rd) Re Rd
Rha+H2O
Mr (Re) – Rha – H2O = Mr (Rd) – Glc – 2H
Glc+H2O

22. PPT-Rha / PPD-Glc (Re и Rd)18
PPT-Rha / PPD-Glc (Re и Rd) Re Rd
1 - M-GlcGlcGlc-H2O+H
2 - M-GlcGlcGlc-2H2O+H
3 - M-GlcGlcGlc-3H2O+H
1 - M-RhaGlcGlc-H2O+H
2 - M-RhaGlcGlc-2H2O+H
3 - M-RhaGlcGlc-3H2O+H
4 - M-RhaGlcGlc-4H2O+H
m/z, Da
m/z, Da

23. Fragmentation Patterns19
Fragmentation Patterns
Sapogenin
Signals, m/z
PPT
459, 441, 423, 405, 367, 349, 281, 229
PPD
443, 425, 407, 369, 351, 283, 231 OT
475, 457, 439, 421, 403, 143, 125, 107
Sapogenin
Signals, m/z
PPT
459, 441, 423, 405, 367, 349, 281, 229
PPD
443, 425, 407, 369, 351, 283, 231 OT
475, 457, 439, 421, 403, 143, 125, 107 OA
457, 439, 393, 249
PPT fragmentation pattern
Relative Intensity, %.
OA sapogenin Da

24. Sapogenin type classification20
Sapogenin type classification
Fig. 1
Fig. 2
Test set of data (17 standard ginsenosides)
OA ginsenosides determined in real samples
Test set of data (17 standard ginsenosides)
PPT, PPD and OT ginsenosides determined in real samples

25. Identification algorithms21
Identification algorithms
Described in literature:
1. Mass spectrum registration in a scan mode (QP, TOF, ion trap, orbitrap, e.t.c.)
2. M calculation using m/z value of [M+Na]+ signal if possible high resolution employed
3. First generation of fragment ions study
4. Sugar chain R1 (R2) composition determination during second and third generation of the fragment ion analysis
5. Sapogenin mass (Ms) determination from the m/z value of the [Sapogenin- H2O+H]+ [Sapogenin-H2O-H]- signal or Ms = M – (MR1 + MR2)
6. Structural elucidation in a form Sapogenin-R1(R2)
In our work:
1. Mass spectrum registration in a LIT-MS scan mode
2. M calculation using m/z value of [M+Na]+ signal
3. Sugar chain R1 (R2) composition determination m/z differences and other features of the fragmentation pattern
4. Sapogenin mass (Ms) determination from the m/z value of the [Sapogenin-H2O+H]+ signal or Ms = M – (MR1 + MR2)
5. Sapogenin fragmentation pattern comparison
6. Structural elucidation in a form Sapogenin-R1(R2)
2 or more MS experiments!

26. Study of the chromatographic behavior of the ginsenosides in a reverse-phase HPLC22

27. Separation of the ginsenosides23
Separation of the ginsenosides
Rg1+Re
Rg1+Re
Acetonitrile, %.
time, min
Acclaim RSLC С18 (150 x 2.1 мм) columns with particle diameters 2.2 µm and 3 µm. Mobile phase: 0.5% HCOOH aueous solution (eluent А), Acetonitrile (eluent B)

28. Separation of the ginsenosides24
Separation of the ginsenosides R1
PPT-20-Glc-6-GlcXyl
Rg1
PPT-20-Glc-6-Glc Re
PPT-20-Glc-6-GlcRha
F11
OT-6-GlcRha
RT5
OT-6-Glc Rf
PPT-20-H-6-GlcGlc
Rh1
PPT-20-H-6-Glc
Rg2
PPT-20-H-6-GlcRha
Rb1
PPD-20-GlcGlc-3-GlcGlc Rc
PPD-20-GlcAra(f)-3-GlcGlc
Rb2
PPD-20-GlcAra(p)-3-GlcGlc
Rb3
PPD-20-GlcXyl-3-GlcGlc F1
PPT-20-Glc-6-H Rd
PPD-20-Glc-3-GlcGlc
Rg3
PPD-20-H-3-GlcGlc
C-K
PPD-20-Glc-3-H
Rh2
PPD-20-H-3-Glc
Intensity, cps.
time, min
Intensity, cps.
time, min
Modified sapogenins < ОТ < PPT < PPD ~ (PPT-H2O) < (PPD-H2O)
GlcGlcGlc < GlcGlc < Glc < Ara(f) < Ara(p) < Xyl < Rha

29. Separation of the ginsenosides25
Separation of the ginsenosides
Ginsenoside
Retention time, min
Resolution
Efficiency,*103 ТP/m
Symmetry R1
8.1
7.50 60
1.0
Rg1
9.5
1.2
130 Re
9.6 36
133
1.1
F11
16.7
0.04
194
RT5
178 Rf
17.0
183
Rg2
19.3
0.14
240
Rh1
1.3
340
Rb1
19.6
4.6
320 Rc
20.5
4.5
530
Rb2
21.4
1.6
290
0.9
Rb3
21.8
0.7
300 F1
22.0
6.0
310 Rd
23.4
40.5
380
Rg3
30.3
21.7
2800
C-K
33.5
4.3
1150
Rh2
34.2 -
1900

30. Optimization of a fast sample preparation procedure for the effective and non-destructive extraction of the ginsenosides 26

31. Ginsenoside extraction27
Ginsenoside extraction
Fig. 1. Ginsenoside Rd content relation with solvent volume used for the extraction. 20 and 30 mL volumes were added by 10 mL portions
Fig. 2. Ginsenoside Rc recovery relation with organic component concentration гинсенозидов (M – methanol, Е – ethanol).
Ginsenoside Rc recovery, %
Ginsenoside Rd, mg/g
330
Organic component content, %
Solvent volume, mL
Final extraction conditions:
Solvent volume - 10 mL
Solvent – H2O:methanol (4:1) mixture
Ultra-sound field 30 min; 30 °C

32. Ginsenoside degradation study28
Ginsenoside degradation study
Ginsenoside content in spiked and non-spiked samples (n=3, P=0.95)
Ginsenoside Sample
Ginsenoside Rg1
Ginsenoside Rb1
Ginsenoside Rc
Peak area, cts. (×107)
R, %
Dry roots
7.9 ± 0.2 -
45 ± 1
9.1 ± 0.2
Spiked dry roots
18.1 ± 0.4 99
50 ± 1 94
14.5 ± 0.3
Spiked model sample
10.9 ± 0.2
107
4.4 ± 0.1 79
4.8 ± 0.1 87
Std. aqueous solution
10.2 ± 0.2
5.5 ± 0.1
330
PPD-20-GlcGlc-3-GlcGlc (Rb1)
PPD-20-GlcAra(f)-3-GlcGlc (Rc)
PPD-20-H-3-GlcGlc (Rg3)
PPD-20-H-3-Glc (Rh2)
(Popovich and Kitts, 2004)

33. Analytical characteristics29
Analytical characteristics
Compound
Detection methods
Linearity, µg/mL
Calibration equation
Correlation coefficient, r2
LOD, µg/mL
Rg1 1
0.02—5
y = 5.0×107x+5.4×106
0.992
0.006 2
y = 4.8×105x+2.0×104
0.999 Rf
0.01—5
y = 10.2×107x+4.5×106
0.003
0.003—5
y = 4.2×106x+1.8×105
0.001 Re
0.03—5
y = 3.2×107x+1.4×106
0.998
0.009
y = 9.0×105x+1.7×104
Rb1
0.04—5
y = 1.4×107x+0.3×106
0.01
y = 4.7×105x–4.3×105
0.996
Detection methods:
Peak area evaluation in scan mode
Peak area evaluation in SIM mode
Sapogenin
SIM Signals, m/z
PPT
459, 441, 423, 405
PPD
443, 425, 407 OT
475, 457, 439, 421, 403

34. Selective method development for pseudoginsenosides RT5 and F11 in MRM mode30

35. Pseudoginsenoside mass spectra31
Pseudoginsenoside mass spectra
F11, ESI(+), MS/MS OT-6-Glc-Rha
M1, M2
Intensity, cps.
m/z, Da
Ginsenoside F11 [M+H]+ m/z=801 fragmentation in ESI(+) mode. M = 800 Da, M1 = 474 Da, M2 = 142 Da.

36. Pseudoginsenoside separation32
Pseudoginsenoside separation
Pseudoginsenoside F11 and RT5 standard mixture chromatogram (200 and 300 ng/mL), in MRM detection mode m/z 801→143 and 655→143, correspondingly.
F11
Intensity, cps.
RT5
Time, min

37. Pseudoginsenoside detection parametersА1
Pseudoginsenoside detection parameters
Parameter
Value
Injected volume
0.020 mL
Temperature
30 °С
Flow
0.40 mL/min
Mobile phase
32% acetonitrile 68% aqueous solution HCOOH (0.5%)
Declustering potential (DP)
50 V
Entrance potential (EP)
7 V
Collision energy (CE)
30 V
MRM for F11 OT-6-GlcRha
801 → → 143
MRM for RT5 OT-6-Glc
655 → → 143
Detection polarity
Positive

38. F11 and RT5 analysis in real objects33
F11 and RT5 analysis in real objects
Table 1. Analytical characteristics of the MRM detection of pseudoginsenoside approach
Sample
Linearity, ng/mL
Calibration equation
Correlation coefficient, r2
LOD, ng/mL
F11
50—2000
y = 7x-56
0.999 20
RT5
30—3000
y = 103x+2040 10
Table 2. Pseudoginsenoside F11 and RT5 content in ginseng samples (N=3, P=0.95)
Sample
F11 content, µg/g
RT5 content, µg/g
Root slices P. quinquefolius Leiyunshang (China)
200±20
< 1
Dry root (Siberia)
76±7
~ 1
Dry root (Bryansk)
< 2

39. Ginseng analysis and method approval34

40. Ginsenosides detected35
Ginsenosides detected
Different sapogenin based ginsenosides
Samples
Ginsenosides
PPT
PPD ОА ОТ
Modified sapogenin
Total
Dry roots (Russian Far East) 12 16 1 30
Fresh Roots (Russian Far East) 5 15 20
Chopped Fresh Root (Russian Far East) 6 18 2 24
Dry Roots (Siberia) 10 14 3 29
Dry Roots (Bryansk) 9 19
Dry Root Slices P. quinquefolius 21
Korean Ginseng Tea
Ginseng Phytoproduct 7 13 4 27
330

41. Ginsenosides detected36
Ginsenosides detected
Samples
Ginsenosides, mg/g
Rg1 Rf
F11 R1 Rd
Rb1
Rb2
Dry roots (Russian Far East)
1.7
1.5
≤ 0.005
0.2
0.3
0.4
Fresh Roots (Russian Far East)
0.1
Chopped Fresh Root (Russian Far East)
0.5
Dry Roots (Siberia)
1.4
0.6
0.7
Dry Roots (Bryansk)
Korean Ginseng Tea
0.05
Dry Root Slices P. quinquefolius
1 mg/g in sample
0.1 g in 10 mL
extraction
10 µg/mL
(0.01—5 µg/mL linearity)
330
Minimal sample preparation

42. Ginsenoside monitoring37
Ginsenoside monitoring
1 peak
1 spectrum

43. Existing identification methods Quality control methods38
Analysis schemes
Developed approach
Existing identification methods
Quality control methods
Extraction
H2O and organic solvent
Fractionating
RP HPLC
Mass spectra registration
MS scanning
Mass spectra registration
Several generations of fragment ions
UV detection
Using Linear Ion Trap
Ginsenoside identification
tR values
Ginsenoside Identification
New structure determination
NMR, e.t.c.
Quantitative analysis

44. 39
Conclusion
1. The rules of the formation of the ginseng saponin mass spectra using electrospray ionization and linear ion trap (LIT) mass spectrometry were established. It is shown that the use of LIT produces highly informative mass spectra containing the characteristic signals of the molecular and fragment ions. 2. An algorithm that allows the identification of structural fragments of the ginsenosides from mass spectrometric data has been developed. The fragmentation patterns for the three main sapogenins (PPT, PPD and OT) were extracted. On the basis of experimental data the fragmentation pattern of oleanolic acid sapogenin has been proposed. 3. The possibility of using the HPLC-MS/MS method for simultaneous determination of PPT, PPD and OT ginsenosides in a gradient mode. Selected conditions of simultaneous determination of 17 ginsenosides with detection limits of 1-10 ng/ml. The high selectivity of the separation in the case of structural isomeric ginsenosides with different sapogenins, and low - in the case of ginsenosides, which differ by the presence of Rha sugar residue. 4. The method of ginsenosides extraction from plant material with methanol:water (1:4) mixture in ultrasonic field. It is shown that during the extraction no analyte degradation is observed. 5. A quick approach for selective HPLC-MS/MS determination of pseudoginsenosides RT5 and F11 in the presence of other ginseng saponins in MRM mode under chosen electrospray ionization conditions. The detection limits in water for RT5 and F11 were 10 and 20 ng/ml, which allows the determination of these compounds in vegetable raw materials and ginseng based products.

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