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RSC Advances
(Cairo, Egypt). Cytomid® tablets labeled to contain 250 mg FLT Where A is the frequency factor, R is the gas constant ¼ 8.314 J
per tablet (product of Cipla Ltd., Mumbai, India) were molꢀ1 Kꢀ1 and T is the absolute temperature (ꢁK ¼ 273 + C).
ꢁ
purchased from a local Egyptian pharmacy. Methanol (HPLC
grade) was obtained from Kanto Chemical Co., INC. (Tokyo,
Japan). Orthophosphoric acid (85% w/v) and potassium dihy-
drogen phosphate were obtained from Wako Pure Chemical
Industries, Ltd. (Osaka, Japan). Hydrochloric acid (35–37%) and
sodium hydroxide were obtained from Chameleon Reagent
(Osaka, Japan). Hydrogen peroxide (30% w/v) was obtained
from Nacalai Tesque, Inc. (Kyoto, Japan). Puried water was
obtained using Millipore direct-Q 3UV water purication system
(Molsheim, France). Aqueous solutions of 0.3 M HCl and 0.03 M
NaOH were prepared. Phosphate buffer of pH 4.0 was prepared
by adjusting the pH of 0.04 M potassium dihydrogen phosphate
with few drops of orthophosphoric acid (85% w/v). Britton–
Robinson buffer (BRB) containing 0.04 M boric acid, 0.04 M
orthophosphoric acid, and 0.04 M acetic acid was prepared and
the pH was adjusted to 2.0–12.0 with 0.2 M NaOH.20
General recommended procedures
Calibration graph. Accurately measured volumes of FLT
standard solution were diluted with the mobile phase to obtain
nal concentrations over the range of 0.2–25.0 mg mLꢀ1. Twenty
mL aliquots were injected (triplicate) and eluted with the mobile
phase under the optimum chromatographic conditions. The
average peak areas of FLT were plotted versus the corresponding
drug concentrations (mg mLꢀ1) and the regression equation was
derived.
Assay of tablets. Ten tablets were accurately weighed, nely
pulverized and thoroughly mixed. An accurately weighed
amount of the powder equivalent to 10.0 mg FLT were trans-
ferred into 100 mL volumetric ask and diluted to the mark
with methanol. The solution was sonicated for 30 min then
ltered. Accurately measured volumes of the ltrate were
diluted with the mobile phase and chromatographed under the
optimum conditions. Nominal contents of FLT were calculated
using the regression equation.
Degradation protocol. 0.5 mL aliquots of FLT standard
solution (200.0 mg mLꢀ1) were transferred into a series of 10 mL
glass vials followed by 2 mL of 0.3 M HCl or 0.03 M NaOH.
Solutions were then incubated in a thermostatically controlled
water bath at different temperature settings (70–100 and
60–90 ꢁC for acidic and alkaline degradation, respectively).
Samples were taken at appropriate times (10–40 min), neutral-
ized and made up to 5.0 mL with the mobile phase and mixed
well. Twenty mL aliquots were injected and eluted under the
optimum chromatographic conditions and the remaining drug
Standard solutions
A stock solution of FLT containing 2000.0 mg mLꢀ1 was
prepared in methanol and a standard solution (200.0 mg mLꢀ1
)
was prepared by dilution with the same solvent. Working
solutions of appropriate concentrations were prepared by
dilution with methanol. The solutions were stable for one week
ꢁ
when stored in a refrigerator at 4 C.
Chromatographic conditions
Separation was performed on a Cosmosil 5C18-MS column (150
mm ꢃ 4.6 mm id, 5 mm particle size) from Nacalai Tesque, Inc.
A mobile phase consisted of methanol–phosphate buffer (0.04
M; pH 4.0) (75 : 25, v/v) was pumped at a ow rate of 1 mL minꢀ1
and UV-detection was set at 240 nm.
concentrations were calculated using the regression equation. A
a
plot of log
versus time (t) was constructed. The observed
a ꢀ x
rst-order degradation rate constants (k) and half-lives (t1/2),
were calculated using eqn (1) and (2), respectively. Arrhenius
plots for acidic and alkaline degradation were constructed by
plotting log k versus 1/T (ꢁK) and the activation energies (Ea) for
FLT degradation were calculated from the slopes according to
eqn (3).
Calculation of degradation kinetics parameters
Kinetics parameters for the degradation of FLT were calcu-
lated.21 The observed rst-order degradation rate constants (k)
were calculated from the slopes of semi-logarithmic plots of
Oxidative stress as well as neutral hydrolysis studies were
also carried out as follow: 2 mL of H2O2 (30 w/v) or distilled
water were added to 0.5 mL of FLT standard solution (200.0
mg mLꢀ1) and heated for 2 h at 80 ꢁC. For the photo-degradation
study; 0.5 mL of FLT standard solution (200.0 mg mLꢀ1) was
transferred into a series of glass vials and diluted with 2 mL of
methanol, water, or methanol–water mixture (1 : 1, v/v), then
exposed to UV-lamp emitting radiation at a wavelength of 254
nm for 24 h. Then, the solutions were made up to 5 mL with the
mobile phase, mixed well and eluted under the optimum
chromatographic conditions.
a
log
versus time (t) in accordance with eqn (1):
a ꢀ x
a
kt ¼ 2:303 log
:
(1)
a ꢀ x
Where (a) is the initial drug concentration and (a ꢀ x) is the
remaining drug concentration.
Half-life times (t1/2), for the rst order degradation reactions,
were calculated according to eqn (2):
0:693
t1=2
¼
:
(2)
k
pH-rate prole for FLT. 1 mL of FLT stock solution (2000.0
mg mLꢀ1) was transferred into a series of 10 mL glass vials, 9 mL
of BRB (pH ¼ 2.0–12.0) were added and mixed well. Solutions
were heated under reux in a boiling water bath. 1 mL aliquots
were taken at appropriate times (10–40 min), neutralized and
diluted to 10 mL with the mobile phase then analyzed. The
Activation energy (Ea) was calculated from the slope of
Arrhenius plot of log k versus 1/T in accordance to eqn (3):
Ea
log k ¼ log A ꢀ
:
(3)
2:303RT
This journal is © The Royal Society of Chemistry 2015
RSC Adv., 2015, 5, 3206–3214 | 3207
El-Shaheny
