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UPLC/ Tandem mass and antimicrobial activity of Khaya senegalensis (A.Juss.)
generally limited to the modification of A and B rings. On
the other hand, Meliaceae limonoids are characterized by
high degree of oxidation and rearrangement exhibited in
the parent limonoid structure (1).
Ultra performance liquid chromatography (UPLC) has
been one of the important physicochemical methods for
identification of constituents in plant extracts, due to its
rapidity, sensitivity, and low levels of the sample needed
(2).
UPLC–UV analysis usually does not provide sufficient
information necessary to distinguish between structural
isomers. UPLC equipped with ESI /MS method could be
useful in the characterization of these compounds. Data
dependent tandem mass spectrometry (MS/MS) was
performed to assist in the structural elucidation of both
aglycones and glycosidic conjugates, as well as the
structures of limonoids.
Anti-microbial has been and will continue to prioritize
the health needs in all communities. Particularly, when we
encounter the emergence of microbes resistance to
antibiotics as result of their overuse. Which makes the
search for safe and effective alternatives as essential needs. using a MicrOTOF-Q hybrid quadrupole time-of-flight
Khaya senegalensis, one of Meliaceae plants, have long
been used in folk medicine. Flowers are used in medicines
against stomach complaints and syphilis. (3). Although,
the chemical composition of leaves, bark and seeds have
been studied (4, 5), according to our knowledge no
phytochemical neither biological studies of flowers were
reported. This study attempt to investigate their flavonoids
and limonoids constituents as well as antimicrobial and
cytotoxic activity.
2. EXPERIMENTAL
2.1. Chemicals and reagents
Acetonitrile and formic acid (LC/MS grade) were
obtained from J. T. Baker (Deventer, The Netherlands),
milliQ water was used for UPLC analysis. Rutin,
isoquercitin, isorhamnetin, kaempferol-3-O-ß-D-
rutinoside, umbelliferone and kaempferol were obtained
from Chromadex (Wesel, Germany). All other chemicals
and standards were provided from Sigma-Aldrich (St
Louis, MO, USA).Sodium dodecyl sulphate (SDS), (3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium
bromide) (MTT), Doxrubicin (DOX) (Pharmacia,
Belgium).
2.2. Plant material
Khaya senegalensis (A. Juss) flowers were collected
during April-May from Giza Zoo, Egypt. Plant specimens
are deposited at Faculty of pharmacy-Cairo University
(voucher No.: 10-6-2014a).
2.3. Plant extraction
The shadow dried powdered flowers (100g) were
defatted with petroleum ether then extracted with acetone,
the acetone was evaporated under vacuum till dryness to
afford brown residue (20gm).
@Real Academia Nacional de Farmacia. Spain
Acetone fraction of defatted powder of Khaya
senegalensis (A. Juss) flowers (20 ± 0.06 mg) was
extracted with 1.8 mL aq.80% MeOH for 10 hours using
an orbital shaker in the dark.1 mg of extract was dissolved
in 1 mL 100% MeOH. The extract was spiked with (20 µg)
umbelliferon as a standard. After sonification for 2 min.,
sample was centrifuged at 13000 rpm for 3 minutes to
remove insoluble material, supernatant was injected.
2.4. High resolution UPLC-PDA -MS analysis
Chromatographic separations were performed on an
Acquity UPLC system (Waters) equipped with a HSS T3
column (100 × 1.0 mm, particle size 1.8 µm; Waters)
applying two elution binary gradients at a flow rate of 150
µL min–1: (1) 0 - 1 min, isocratic 95% A (water/formic
acid, 99.9/0.1 (v/v)), 5% B (acetonitrile/formic acid,
99.9/0.1 (v/v)); 1 - 16 min, linear from 5 to 95% B; 16 t-
18 min, isocratic 95% B; 18 - 20 min, isocratic 5% B. The
second binary eluent (2) was composed of ammonium
acetate 50 mM buffer adjusted to pH 5(A) and 100%
acetonitrile (B) using the same elution gradient as above.
The injection volume was 3.1 µL (full loop injection).
Eluted compounds were detected from m/z 100 to 1000
mass spectrometer (Bruker Daltonics) equipped with an
Apollo II electrospray ion source negative ion mode using
the following instrument settings: nebulizer gas, nitrogen,
1.6 bar; dry gas, nitrogen, 6 L min–1, 190°C;
capillary,5500 V (+4000 V); end plate offset,500 V; funnel
1 RF, 200 Vpp; funnel 2 RF, 200 Vpp; in-source CID
energy, 0 V; hexapole RF, 100 Vpp; quadrupole ion
energy,5 eV; collision gas, argon; collision energy, 10 eV;
collision RF200/400 Vpp (timing 50/50); transfer time, 70
µs; prepulse storage, 5 µs; pulser frequency, 10 kHz;
spectra rate, 3 Hz. Internal mass calibration of each
analysis was performed by infusion of 20µL 10 mM
lithium formiate in isopropanol/water, 1/1 (v/v), at a
gradient time of 18 minutes using a diverter valve.
Compounds were characterized by their UV-vis. spectra
(220-600 nm), retention times relative to umbelliferone,
mass spectra and comparison to our in-house database and
reference literature (6).
2.4. Tandem mass spectrometry (MS–MS)
MicrOTOF-Q: Precursor ions were selected and
fragmented in the collision cell applying collision energies
in the range of 10–30 eV. Argon was used as collision gas.
Product ions were detected using the following parameter
settings: pulser frequency, 10 kHz; spectra rate, 1.5 Hz.
For CID of in-source fragment ions, in-source CID energy
was increased from 0 to 100 V. Ion Trap MS: MSn mass
spectra were also obtained from a LCQ Deca XP MAX
system (ThermoElectron, San Jose, USA) equipped with a
ESI source (electrospray voltage 4.0 kV, sheath gas:
nitrogen; capillary temperature: 275 °C) in negative
ionization modes. The Ion Trap MS system is coupled with
the exact Waters UPLC setup and using same elution
gradient. The MSn spectra were recorded by using the
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