LCMS PHYTOCHEMICAL ANALYSIS

Phytochemical Analysis using LCMS

1. LIQUID CHROMATOGRAPHY MASS SPRCTROMETRY PHYTOCHEMICAL ANALYSIS DEPARTMENT OF PHARMACOGNOSY DELHI PHARMACEUTICAL SCIENCES AND RESEARCH UNIVERSITY SUBMITTED BY BUSHRA 64/MPH/DPSRU/2019 M.PHARM 1 ST SEMESTER

2. CONTENT Introduction to LC & MS Integrating LCMS Application of LCMS Phyto -chemical Analysis of Solanum Plant Ptochemical analysis of Methanol Extracts of Seed Shells of Archidendron bubalinum References

3. LIQUID CHROMATOGRAPHY MASS SPECTROMETRY Combination of chromatography & mass spectrometry. Analytical technique that combines physical separation of liquid chromatography with mass analysis capabilities of mass spectrometry . Capable of providing structure, molecular weight, empirical formula, & quantitiative analysis of an analyte. Technique through which a mixture is being separated between two phases i.e. stationary phase & mobile phase.

4. LIQUID CHROMATOGRAPHY Technique through which a mixture is being separated between two phases i.e. Stationary phase Mobile phase Liquid or solid Liquid Liquid chromatography involves following techniques: Paper chromatography Thin layer chromatography High performance/ pressure liquid chromatography Adsorption column chromatography Ion exchange chromatography Liquid liquid chromatography

5. Instrumentation of liquid chromatography (specifically HPLC): Solvent reservoir* Degasser Pump* Pre-guard column Analytical column Detector* Recorder Solvent reservoir: selection based on elution technique i.e. Isocratic elution Gradient elution Types of Pump are: Reciprocating pumps Single piston pumps Dual piston pumps Pneumatic pumps

6. Detectors for LC Systems MASS SPECTROMETRY FLURESCENCE UV-VIS COMDUCTIVITY EVAPORATIVE LIGHT SCATERRING REFRACTIVE INDEX INCREASING SENSITIVITY When analyte passed through detector, a change (e.g. increase or decrease) in the optical property will be observed and recorded. Chromatograms obtained using these optical detectors primarily identify or qualify substances based on the retention time and quantitate substances based on the peak area and intensity.

7. APPLICATIONS OF LIQUID CHROMATOGRAPHY SEPERATION PURIFICATION QUANTIFICATION

8. MASS SPECTROMETRY An instrumental technique in which sample is converted to rapidly moving positive ions by electron bombardment & charged particles are separated according to their mass. Record of mass spectroscopy/spectrometry is called Mass Spectra Defined as a plot of relative abundance against ratio of mass to charge (m/z). Mass Spectra is useful for following: to establish structure of new compounds To give molecular formula To give exact molecular formula To indicate presence of functional group . MASS SPECTROSCOPY : A qualititative & quantitative analytical technique through which a molecular formula/mass can be detected.

9. Components of Mass Spectrometry

10. IONIZATION SOURCES Electron impact (EI), Chemical ionization (CI), Desorption ionization (DI), Matrix-assisted laser desorption/ionization (MALDI), Desorption electro spray ionization (DESI), Electro spray ionization (ESI), and Atmospheric pressure chemical ionization(APCI ). TYPES OF DETECTORS IN MS Electron multipliers Photo multipliers Photographic method Scintillation (e.g. DELAY detector)

11. WORKING OF MASS SPECTROMETER Stage 1: Ionisation Stage 2: Acceleration Stage 3: Deflection Stage 4: Detection

12. INTEGRATING LCMS Technique that combines physical separation of liquid chromatography with mass analysis capabilities of mass spectrometry . PRINCIPLE : A method that combines separation power of HPLC and detection power of MASS SPECOTRMETRY

13. LC UNIT Solvent reservoir Degasser Pump Sample inlet Pre-guard column column INTERFACE BETWEEN LC & MS IONIZATION SOURCE USUALLY ESI ION GUIDE Electrostatic Lens that efficiently introduces the ions into the MS Mass analyzer unit DETECTORS Components of LCMS system

14. In LC-MS the detector from the LC column is removed & fits the column to the INTERFACE INTERFACE Connects an HPLC system to a mass spectrometer. basic requirements for a successful interface : maintaining chromatographic performance (minimum additional peak broadening), high transfer efficiency from LC to MS, and No degradation in mass spectrometric performance main challenge in LC/MS interfaces : high liquid flows from HPLC make it very difficult to maintain the high vacuum required for the function of a MS number of different LC/MS interfaces to overcome the difficulty : Direct Liquid Introduction Moving Belt System Thermospray Continuous-Flow FAB

15. Common Interfaces: Electrospray (ESI) Atmospheric Pressure Chemical Ionization (APCI) Special Interfaces: Particle Beam. Atmospheric Pressure Photoionization

16. Schematic diagrams of LCMS system

17. Applications of LC-MS Characterization of Impurities and Decomposition Products in Bulk drug substances Recently, Feng et al. investigated oxidative degradation products of an antifungal agent, SCH 56592 (Scheme 7), by both LC/MS and LC/NMR. High-Throughput LC/MS for Combinatorial Chemistry Molecular weight determination Structure elucidation Pharmacokinetic Studies of Drugs . LC/MS and LC/MS/MS become most widely used techniques to determine drug concentrations in biological matrices. Identification of Drug Metabolites . Recently, Hop et al. used combination of LC/MS, LC/MS/MS, and NMR techniques to identify metabolites of a substance P ( Neurokinin 1 receptor) antagonist, compound A (Scheme 10), in rat hepatocytes and rat plasma

18. Family Solanaceae presence of various photochemical in Solanum plant use in medicine and in food as functional foods or dietary supplements S. corymbiflorum leaves Phenolic compounds : chlorogenic acid, caffeic acid, gallic acid Flavonoids Alkaloids S. nigrum L. leaves, fruits, and stem tannins, flavanoids , alkaloids, glycosides, steroids PHYTOCHEMICAL ANALYSIS of SOLANUM PLANT

19. Quadrapole time-of-flight mass spectrometry ( qTOF -MS) fingerprinting method Improved methods to generate stable and reproducible results. To investigate the phyto -chemical composition of S. retroflexum leaf extracts with the aid of UPLC- qTOF -MS . Plant Collection: Solanum retroflexum leaves Sep tember and October 2015 leaves air-dried under shade and stored in airtight containers at room temperature Materials and Methods:

20. Extraction of Metabolites : different sample extracts obtained by using aqueous methanol ( 40%, 60%, and 80%, methanol/ water, v/v) grounded (2.0 g) leaves of S. retroflexum extracted with different concentrations of aqueous methanol and sonicated at room temperature for 15 minutes . resultant mixture centrifuged for 10 minutes and evaporated using a Buchi rotary evaporator . extracts dried to completeness overnight, dissolved again in methanol, and filtered through 0.22 μm syringe filters and kept at −20 ° C prior to analysis

21. H igh-Performance Liquid Chromatography and Mass Spectrometry : mobile phase : solvent A - formic acid (0.1%) in deionised water solvent B - acetonitrile with 0.1% formic acid gradient elution method - 30 min Initial conditions : 98% A at a flow rate of 0.4 mL /min - constant for 1 min. Conditions changed to 97% A at 3 min, Reduced slightly to 92% A at 4 min - for 21 min, changed to 50% A at 25 min. Conditions were changed to 5% A at 26 min - for 2 min returned to initial conditions of 98% A at 28 min - constant 2 min to allow re-equilibration before the next run Elution monitored using a photodiode-array detector (PDA) collecting 20 spectra per second between the 200 and 500nm range.

22. MS data acquired using both positive and negative ESI modes. After series of optimization, the following settings found to be optimal: capillary voltage of 2.5 kV, sample cone potential of 30 V, source temperature of 120°C, desolvation temperature of 450°C, cone gas flow of 50 L/h and desolvation gas flow of 550 L/h, and multichannel plate detector potential of 1600 V. Efficient fragmentation during identification , data collected using collision energy ramp of 10–30 eV . * higher collision energy ramp of 165–60 eV ( if necessary)

23. 4. Results and Discussion: Methanol Extraction of Phytochemicals methanol extracts of Solanum retroflexum generated using the different concentrations of aqueous methanol. A total of 30 metabolites identified - chlorogenic acids (CGAs), flavonoids , and alkaloids. Chlorogenic Acids Characterisation of Caffeoylquinic Acids . Molecules 1, 2 & 3 were identified as 3CQA, 4CQA & 5CQA Characterisation of Feruloylglycoside molecular weight 356; m/z 355; 1 molecule identified - base peak at m/z 175 and 160 Characterisation of Di- caffeoylquinic Acid and Caffeoylquinic Acid- glucoside di-caffeoylquinic acid and caffeoylquinic acid glucoside were identified - ion at m/z 515 7–13 molecules identified as di -CQA or CQA glycoside. Characterisation of p- Coumaroylquinic Acid and Coumaroyl-hexose ion peaks identified - 3- pCoQA ( 14), trans 5-pCOQA (15), cis-5-pCOQA ( 16), and 4-p-COQA (18). Molecule 17 - Coumaroyl-hexose m/z 339

24. Characterisation of Feruloylquinic Acids: peaks of feruloylquinic acids trans-5-FQA ( 19), cis-5-FQA (20), 4-FQA ( 21), 3-FQA (22) Characterisation of Flavonoid Derivatives: total of six flavonoids 23, 24, 25, 26, 27, and 28 detected Characterisation of Alkaloid Derivatives : Molecules 29 & 30 m/z 884 & 868 as solasonine & solamargine

25. LC-MS Studies on Methanol Extracts of Seed Shells of Archidendron bubalinum Archidendron bubalinum belongs to the family Fabaceae or Leguminosae . The species is indigenous to Sumatra in Indonesia, Peninsular Malaysia and Thailand. Objective : to identify the types of compounds found in the methanol extracts from seed shells of Archidendron bubalinum from Lampung Indonesia. Figure 1 : A) Fruits, B) Fruit shells, C) Seeds, D) Seed shells of Archidendron bubalinum .

26. MATERIALS AND METHODS : seeds of Archidendron bubalinum collected peeled to separate the seed shells seed shells thoroughly washed and sun dried for 4 h. dried seed shells soaked in methanol (1:10 (w/v)) and extracted by maceration for 24 h. supernatant filtered. Soaking process repeated once again in the same sample and supernatant was filtered. supernatant collected together and concentrated using rotary evaporator at 50 degree C.

27. Liquid Chromatography-Mass Spectrometry (LC-MS) studies Methanol extracts chemical constituents determined. HPLC interfaced with a Q-TOF mass spectrometer fitted with an ESI. HPLC column : Phenomenex 5μ C8, (150 × 2 mm i.d .) Solvent : methanol with 0.3% formic acid. flow rate - 0.1 mL /min. isocratic elution. The MS spectra acquired in the positive ion mode. drying gas (N2) – temperature - 350 degree C; flow rate of 6 mL /min, nebulizing pressure - 25 psi. Sample injection : 0.5 g of sample extracts diluted with methanol filtered with 0.22 μm nylon filter prior to analysis. A 5 μl volume of the extracts injected for analysis. The mass fragmentations identified.

28. Figure 2 : LC-MS chromatogram of methanol extract of Archidendron bubalinum seed shells

29. Figure 3 : Mass spectrums of 5 components of methanol extracts.