Stability-indicating HPLC/FL detection method for agomelatine
mL volumetric flasks to obtain final concentrations of 0.4–40.0
ng/mL. Accurately measured volumes of IS were added to obtain
fixed final concentration of 20.0 ng/mL. Volumes were com-
pleted with the mobile phase and the solutions were mixed well.
Twenty-microliter aliquots were injected (triplicate) and eluted
with the mobile phase under the optimum chromatographic
conditions. The average peak area ratios of AGM/IS were plotted
against the corresponding drug concentrations (ng/mL) and the
regression equation was derived.
removed from the water bath and cooled. The solution was
transferred into 25 mL volumetric flasks, completed to
volume with water and mixed well. An aliquot of 0.1 mL of
the solution was quantitatively transferred into a 10 mL volu-
metric flask and an accurately measured volume of IS was
added to obtain a fixed final concentration of 20.0 ng/mL.
The solution was diluted to volume with the mobile phase
and mixed well. Twenty-microliter aliquots were injected (trip-
licate) and eluted with the mobile phase under the optimum
chromatographic conditions.
Assay of tablets. Ten tablets were accurately weighed, finely
pulverized and thoroughly mixed. An accurately weighed
amount of the powder equivalent to 10.0 mg of AGM was
transferred into 100 mL volumetric flask; the volume was com-
pleted with methanol, sonicated for 30 min and filtered with
Photolytic degradation. Accurately measured volumes of AGM
standard solution (equivalent to 100.0 μg) were transferred into
three 25 mL volumetric flasks. The solutions were diluted to vol-
ume with distilled water, methanol and a distilled water– meth-
anol mixture (1: 1, v/v), respectively. Solutions were exposed to
UV irradiation at 254 nm at a distance of 15 cm from the light
source for 6 days. At the specified times, the flasks were re-
moved from the UV chamber. Aliquots of 0.1 mL of each
solution were quantitatively transferred into 10 mL volumetric
flasks and an accurately measured volume of IS was added to
obtain a fixed final concentration of 20.0 ng/mL. Solutions
were diluted to volume with the mobile phase and mixed
well. Twenty-microliter aliquots were injected (triplicate) and
eluted with the mobile phase under the optimum chromato-
graphic conditions.
0
.45 μm cellulose acetate disposable syringe filters. Appropri-
ate volumes of the filtrate were successively diluted with the
mobile phase to obtain sample solutions of concentrations
within the working range and the procedure described for
the ‘calibration graph’ was followed. Nominal contents of
the tablets were calculated using the previously derived
regression equation.
Procedures for stability studies
Acidic and alkaline degradation. Accurately measured volumes
of AGM standard solution (equivalent to 100.0 μg) were trans-
ferred into two series of small conical flasks. To the first series,
4
.0 mL of 2.0 M NaOH were added, whereas 4.0 mL of 4.0 M
HCl were added to the second series. The solutions were heated
in a boiling water bath for increasing intervals (10-40 min). At the
specified times, the flasks were removed from the water bath,
cooled and neutralized to pH 7 with either 2.0 M HCl or 4.0 M
NaOH, respectively. Solutions were transferred into 25 mL volu-
metric flasks, completed to volume with water and mixed well.
Aliquots of 0.1 mL of each solution were quantitatively trans-
ferred into 10 mL volumetric flasks and an accurately measured
volume of IS was added to obtain a fixed final concentration of
In vitro analysis of AGM in spiked human plasma
The extraction procedure described by Patil et al. (5) was
followed. Aliquots of 1.0 mL of human plasma were transferred
into a series of 5-mL screw-capped centrifugation tubes. Plasma
samples were spiked with increasing volumes of AGM standard
solution followed by 200 μL of IS solution. The samples were
extracted by vortex mixing with 3 mL of ethyl acetate for 5
min then centrifuged at 3500 rpm for 15 min. Ethyl acetate
layers were collected into small beakers and evaporated to
dryness at room temperature. The dried residues were
reconstituted with 2.0 mL of the mobile phase (final concentra-
tion range: 1.0–40.0 ng/mL) and filtered through a 0.45-μm
cellulose acetate disposable syringe filter. Twenty-microliter
aliquots were injected (triplicate) and eluted with the mobile
phase under the optimum chromatographic conditions. A
calibration graph was constructed by plotting the peak area
ratio (AGM/IS) against concentration (ng/mL) and the regres-
sion equation was derived.
2
0.0 ng/mL. Solutions were diluted to volume with the mobile
phase and mixed well. Twenty-microliter aliquots were injected
triplicate) and eluted with the mobile phase under the optimum
(
chromatographic conditions.
Neutral degradation. An accurately measured volume of AGM
standard solution (equivalent to 100.0 μg) was transferred into
a small conical flask, and 4.0 mL of distilled water were added.
The solution was heated in a boiling water bath for 1 h. At the
specified time, the flask was removed from the water bath and
cooled. The solution was transferred into a 25 mL volumetric
flask, completed to volume with water and mixed well. An ali-
quot of 0.1 mL of the solution was quantitatively transferred into
In vivo analysis of AGM in plasma from a human volunteer
1
0 mL volumetric flask and an accurately measured volume of IS
A blank blood sample was withdrawn from the volunteer before
oral administration of a single dose of InspagoW tablets
containing 25 mg AGM. Five-milliliter blood sample was
was added to obtain a fixed final concentration of 20.0 ng/mL.
The solution was diluted to volume with the mobile phase and
mixed well. Twenty-microliter aliquots were injected (triplicate)
and eluted with the mobile phase under the optimum chro-
matographic conditions.
2
collected into spray-coated K EDTA tube 1.5 h after administra-
tion of the tablet. Plasma samples were immediately separated
by centrifugation at 3500 rpm for 15 min and kept frozen at -
20°C until analyzed after gentle thawing. Aliquots of 1.0
mL of human plasma were transferred into 5-mL screw-
capped centrifugation tubes, and 200 μL of IS solution was
added. The same extraction procedure described for ‘In vitro
analysis of AGM in spiked human plasma’ was followed. The
Oxidative degradation. Accurately measured volume of AGM
standard solution (equivalent to 100.0 μg) was transferred into
a small conical flask. 4.0 mL of H O solution (30%, w/v) were
2 2
added. The solution was heated in thermostatically controlled
water bath at 80°C for 1 h. At the specified time, the flask was
Luminescence 2014
Copyright © 2014 John Wiley & Sons, Ltd.
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