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This publication discusses the use of a linear ion trap for rapid ginsenoside profiling in roots and ginseng-based products. The study aims to develop new methods for analyzing ginseng ingredients and evaluate the analytical capabilities of these approaches.

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About OMICS Group

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  1. About OMICS Group OMICS Group International is an amalgamation of Open Access publications and worldwide international science conferences and events. Established in the year 2007 with the sole aim of making the information on Sciences and technology ‘Open Access’, OMICS Group publishes 400 online open access scholarly journals in all aspects of Science, Engineering, Management and Technology journals. OMICS Group has been instrumental in taking the knowledge on Science & technology to the doorsteps of ordinary men and women. Research Scholars, Students, Libraries, Educational Institutions, Research centers and the industry are main stakeholders that benefitted greatly from this knowledge dissemination. OMICS Group also organizes 300 International conferences annually across the globe, where knowledge transfer takes place through debates, round table discussions, poster presentations, workshops, symposia and exhibitions.

  2. About OMICS Group Conferences OMICS Group International is a pioneer and leading science event organizer, which publishes around 400 open access journals and conducts over 300 Medical, Clinical, Engineering, Life Sciences, Phrama scientific conferences all over the globe annually with the support of more than 1000 scientific associations and 30,000 editorial board members and 3.5 million followers to its credit. OMICS Group has organized 500 conferences, workshops and national symposiums across the major cities including San Francisco, Las Vegas, San Antonio, Omaha, Orlando, Raleigh, Santa Clara, Chicago, Philadelphia, Baltimore, United Kingdom, Valencia, Dubai, Beijing, Hyderabad, Bengaluru and Mumbai.

  3. Lomonosov Moscow State University 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 - 2014

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

  5. 2 Isomeric ginsenosides • 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. 3 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. 4 Ginsenosidetrasformation W.-Z. Yang et al.Analytica Chimica Acta 739 (2012) 56 t °C (Du X.W. et al.,2004) Soxhlet - H2O (Wang C.Z. et al.,2007) Rg3 Rk1 437 / 623

  8. 4 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. 7 Ionization and fragmentation process study of the ginsenosides

  12. 8 Ginsenoside mass spectra [M-H]- Rb1, ESI(-), Q1 scanPPD-20-Glc-Glc-3-Glc-Glc [M+2H2O-H]- Intensity, cps. [M-Glc-H]- [M-GlcGlc-H]- m/z, Da

  13. 9 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. 10 Pseudoginsenoside mass spectra RT5, ESI(-), MS/MS (CE -50 V) OT-6-Glc [M-H]- Intensity, cps. m/z, Da

  15. 11 Ginsenoside mass spectra Rс, ESI(+), Q1scan 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-LITMSscan mode(LIT – Linear Ion Trap)

  16. 12 Ginsenoside mass spectra Rg1, ESI(+), LITscan Rg1, ESI(+), Q1scan [M+Na]+ <0.5 Da [M+Na]+ m/z, Да Rg1, APCI(+), LITscan m/z, Да CE? DP = 70 V, EP = 10 V, CE = 5 V m/z, Да

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

  18. 14 Ginsenoside mass spectra features 3 Glc+H2O 4 Rg1 Intensity, cps. 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+K M-Glc-3H2O+H m/z, Da

  19. 15 Substituent position isomers Rh1 F1 M-H2O+H M-2H2O+H M-H2O+H m/z, Da m/z, Da

  20. 16 Sugar moieties position isomers Intensity, cps. Rg1 m/z, Da Intensity, cps. Rf M-2H2O+H m/z, Da

  21. 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. 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. 19 Fragmentation Patterns PPT fragmentation pattern Relative Intensity, %. OA sapogenin Da

  24. 20 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. 21 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 chainR1 (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]- signalorMs= M – (MR1 + MR2) 6.Structural elucidation in a formSapogenin-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 chainR1 (R2) composition determinationm/z differencesand other features of the fragmentation pattern 4.Sapogenin mass (Ms) determination from the m/z value of the [Sapogenin-H2O+H]+ signalorMs= M – (MR1 + MR2) 5. Sapogenin fragmentation pattern comparison 6.Structural elucidation in a formSapogenin-R1(R2) 2 or more MS experiments!

  26. 22 Study of the chromatographic behavior of the ginsenosidesin a reverse-phase HPLC

  27. 23 Separation of the ginsenosides Rg1+Re Rg1+Re Acetonitrile, %. time, min AcclaimRSLC С18 (150x 2.1мм) columns with particle diameters 2.2 µm and 3 µm. Mobile phase:0.5% HCOOH aueous solution (eluent А), Acetonitrile (eluent B)

  28. 24 Separation of the ginsenosides 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. 25 Separation of the ginsenosides

  30. 26 Optimization of a fast sample preparation procedurefor the effective andnon-destructive extraction of the ginsenosides

  31. 27 Ginsenoside extraction Fig. 1. Ginsenoside Rd content relation with solvent volume used for the extraction. 20 and 30 mL volumes were addedby 10 mL portions Fig. 2.Ginsenoside Rcrecovery relation with organic component concentration гинсенозидов (M – methanol, Е – ethanol). Ginsenoside Rc recovery, % Ginsenoside Rd, mg/g 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. 28 Ginsenoside degradation study Ginsenoside content in spiked and non-spiked samples (n=3, P=0.95) PPD-20-H-3-GlcGlc (Rg3) PPD-20-H-3-Glc (Rh2) PPD-20-GlcGlc-3-GlcGlc (Rb1) PPD-20-GlcAra(f)-3-GlcGlc (Rc) (Popovich and Kitts, 2004)

  33. 29 Analytical characteristics • Detection methods: • Peak area evaluation in scan mode • Peak area evaluation in SIM mode

  34. 30 Selective method development for pseudoginsenosides RT5 andF11 in MRM mode

  35. 31 Pseudoginsenoside mass spectra F11, ESI(+), MS/MSOT-6-Glc-Rha M1, M2 Intensity, cps. m/z, Da GinsenosideF11 [M+H]+m/z=801 fragmentation inESI(+) mode. M = 800 Da, M1 = 474 Da, M2 = 142 Da.

  36. 32 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. А1 Pseudoginsenoside detection parameters

  38. 33 F11 andRT5analysis in real objects Table 1. Analytical characteristics of the MRM detection of pseudoginsenoside approach Table 2. Pseudoginsenoside F11 and RT5 content in ginseng samples (N=3, P=0.95)

  39. 34 Ginseng analysis and method approval

  40. 35 Ginsenosides detected Different sapogenin based ginsenosides

  41. 36 Ginsenosides detected 1 mg/g in sample 0.1 g in 10 mL extraction 10 µg/mL (0.01—5 µg/mL linearity) Minimal samplepreparation

  42. 37 Ginsenoside monitoring 1 peak 1 spectrum

  43. 38 Analysis schemes Developedapproach Existing identificationmethods Quality controlmethods 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.

  45. Lets Meet again at Pharmacognosy-2015 3rd International Conference and Exhibition on Pharmacognosy, Phytochemistry and Natural Products October 26-28, 2015 Hyderabad, India Theme: Advanced trends for the future of Herbal Drugs and Products Website:http://pharmacognosy-phytochemistry-natural-products.pharmaceuticalconferences.com/

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