Phytochemical Analysis using LCMS

1. DEPARTMENT OF PHARMACOGNOSY
DELHI PHARMACEUTICAL SCIENCES AND RESEARCH UNIVERSITY
SUBMITTED BY
BUSHRA
64/MPH/DPSRU/2019
M.PHARM 1ST SEMESTER

2. 1) Introduction to LC & MS
2) Integrating LCMS
3) Application of LCMS
4) Phyto-chemical Analysis of Solanum Plant
5) Ptochemical analysis of Methanol Extracts of Seed Shells of
Archidendron bubalinum
6) References

3. 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. 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

5. 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

6. 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

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

8. Schematic diagrams of LCMS system

9. Applications of LC-MS
1) 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.
2) High-Throughput LC/MS for Combinatorial Chemistry
 Molecular weight determination
 Structure elucidation
3) Pharmacokinetic Studies of Drugs.
LC/MS and LC/MS/MS
become most widely used
techniques to determine drug concentrations in biological matrices.
4) 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

10.  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

11. 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.
1. Plant Collection:
•Solanum retroflexum leaves
•September and October 2015
•leaves air-dried under shade and
•stored in airtight containers at room temperature
Materials and Methods:

12. 2. 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

13. 3. High-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.

14. 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)

19. 4. Results and Discussion:
1. 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.
2. Chlorogenic Acids
3. Characterisation of Caffeoylquinic Acids.
Molecules 1, 2 & 3 were identified as 3CQA, 4CQA & 5CQA
4. Characterisation of Feruloylglycoside
molecular weight 356; m/z 355; 1 molecule identified - base
peak at m/z 175 and 160
5. 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.
6. 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

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

21. 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.

22. 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.

23. 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.

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

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

27. RESULTS AND DISCUSSION :
Detected 5 peaks - retention time 0.41, 1.28, 1.90, 2,63, and 2.80 minutes (Fig 2).
Each peak fragmented, resulting 5 fragmentation spectrum with candidates mass
(m/z) 365, 436, 436, 247 and 450 (Fig 3)
Results of spectrum interpretation on methanol extracts indicating that there are
substance of phlorizin and astilbin - retention time of 1.90 and 2.80.
These results were confirmed by each of the fragmentation pattern as fig-4 & fig-5
Fig 4: Fragmentation pattern of phlorizin. Fig 5: Fragmentation pattern of astilbin.

28.  Hanafi1, Irawan C, Rochaeni H, Sulistiawaty L, Roziafanto A, SupriyonO
Phytochemical Screening, LC-MS Studies and Antidiabetic Potential of
Methanol Extracts of Seed Shells of Archidendron bubalinum (Jack) I.C.
Nielson (Julang Jaling) from Lampung, Indonesia; Pharmacogn J. 2018
 Daji G, Steenkamp P ,Madala N ,and Dlamini B; Phytochemical
Composition of Solanum retroflexum Analysed with the Aid of Ultra-
Performance Liquid Chromatography Hyphenated to Quadrupole-Time-of-
Flight Mass Spectrometry (UPLC-qTOF-MS); Hindawi, Journal of Food
Quality, Volume 2018.
Chen G, Zhang L, and Pramanik BN; LC/MS: THEORY,
INSTRUMENTATION, AND APPLICATIONS TO SMALL MOLECULES;
SHIMADZU EXCELLENCE IN SCIENCE; Liquid Chromatography
Mass Spectrometry (LCMS).
REFERENCES