Forced Degradation Study on Gliclazide and

Application of Validated Stability-Indicating

HPLC-UV Method in Stability Testing of
Gliclazide Tablets
2007, 66, 751–755

Gulshan Bansal1,&, Manjeet Singh1, Kaur Chand Jindal2

1
Department of Pharmaceutical Sciences and Drug Research, Punjabi University, Patiala 147 002, Punjab, India;
E-Mail: gulshanbansal@rediffmail.com
2
M/S Panacea Biotec Limited, Lalru 140 501, Punjab, India

Received: 9 May 2007 / Revised: 31 July 2007 / Accepted: 3 August 2007

Online publication: 7 September 2007

degradation study on the drug substance

to generate information on degradation
Abstract products that can form under the influence
of hydrolytic, oxidative, dry heat and
Forced degradation study on gliclazide was conducted under the conditions of hydrolysis, photolytic conditions.
oxidation, dry heat and photolysis and an isocratic stability-indicating HPLC-UV method was Gliclazide is an oral hypoglycaemic
developed and validated. All the seven degradation products (I–VII) formed under different agent which lowers the blood glucose
conditions were optimally resolved on a C18 column with mobile phase composed of 40% level by stimulating the pancreatic b-cells
acetonitrile and 60% ammonium acetate solution (0.025 M, pH 3.5) at a flow rate of 0.25 mL to secrete insulin [2]. Chemically, it is
min 1 using 235 nm as detection wavelength. The method was linear between 5–500 lg mL 1 1-(3-azabicyclo[3.3.0]oct-3-yl)-3-(p-tolyl-
drug concentrations. The %RSD of intra- and inter-day precision studies was <1 and <2% sulfonyl)urea (Fig. 1) and the presence of
respectively. Excellent recoveries (99.81–100.97%) proved the method sufficiently accurate. sulfonylurea moiety makes it susceptible
Each peak resolved always with a resolution of >1.90 indicating the method to be rugged to hydrolytic and photolytic degradation
enough. The method was used to study the drug degradation behaviour under the forced [3,4]. However, there is only one report,
conditions. Four degradation products (I–IV) were formed in 0.1 N HCl and water whereas where 4-methylbenzenesulfonamide and
only I and III were formed in 3% H2O2. Two new products V and VI in addition to I, III and IV N-aminocyclopentanopyrrolidine are re-
were formed in 0.1 N NaOH. The drug was stable to thermal and photolytic decomposition. ported as degradation products of glic-
The degradation behaviour in water and 0.1 N NaOH was similar under dark and light lazide in strong acidic medium [5]. In
conditions but a new product VII was formed in 0.01 N HCl in light. In general, the rate of European Pharmacopoeia [6], seven
degradation was accelerated by the light. The method was applied successfully in stability impurities and related substances includ-
testing of gliclazide tablets. ing 4-methylbenzenesulfonamide are lis-
ted in the drug substance monograph.
However, till date no report on the forced
degradation study on gliclazide under the
Keywords conditions of hydrolysis (across wide pH
range), oxidation, dry heat and photoly-
Column liquid chromatography
sis is available. Further, extensive litera-
Stability-indicating
ture review revealed that several
Forced degradation
analytical methods are available for the
Gliclazide
Validation analysis of gliclazide in formulations, in
biological fluids and in the presence of
other antidiabetic agents [7–26]. An
HPLC method is also mentioned in the
European Pharmacopoeia [6] for deter-
Introduction the use of a validated stability-indicating mination of only one related substance,
assay method (SIAM) for stability testing but no single analytical method is avail-
The International Conference on Harmo- of a drug substance or product [1]. It also able for quantification of gliclazide in the
nization (ICH) guidelines Q1AR2 require emphasizes on the conduct of a forced presence of its degradation products.

Full Short Communication Chromatographia 2007, 66, November (No. 9/10) 751
DOI: 10.1365/s10337-007-0394-4
0009-5893/07/11
2007 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH

Fig. 1. Chemical structure of gliclazide
The present study reports the devel-
opment and validation of a stability-indi-
cating HPLC-UV method for gliclazide
based on the information generated by
forced degradation study under ICH
prescribed conditions. The method sepa-
rates all the possible degradation products
of gliclazide in a single run and also
quantifies the drug in the presence of its
degradation products with excellent accu-
racy and precision. The method has been
applied to stability testing of commer-
cially available gliclazide tablets.
Experimental
Chemicals and Reagents
Gliclazide was supplied by Panacea Bio-
tec (India) as a gift sample. Acetonitrile
and methanol (HPLC grade), hydro-
chloric acid, sodium hydroxide pellets,
hydrogen peroxide solution, acetic acid
glacial, ammonium acetate, sodium di-
hydrogen phosphate and orthophos-
phoric acid (all analytical reagent grade)
were purchased from Ranbaxy Fine
Chemicals (India). HPLC grade water
was produced in the laboratory using a
glass triple-distillation assembly (Perfit,
India). The gliclazide tablets were pur-
chased from the pharmacy store.
Instruments
High precision water bath and hot air
oven (Narang Scientific Works, India)
capable of controlling the temperature
with in ±1 and ±2 C were used for the
hydrolytic and thermal degradation
studies, respectively. Photodegradation
was carried out in a photostability
chamber (KBF 240, WTB Binder,
Germany) equipped with a light bank
consisting of two UV (OSRAM L73) and
four fluorescent (OSRAM L20) lamps
and capable of controlling temperature
and humidity in the range of ±2 C and
±5% RH, respectively. The light system
752
complied with option 2 of the ICH
guideline Q1B [27]. At any given time,
UV energy and visible illumination were
tested with a calibrated radiometer (206,
PRC Krochmann GmbH, Germany) and
a calibrated lux meter (ELM 201, Escorp,
India). The HPLC system consisted of
binary pump (515), dual wavelength
detector (2487), rheodyne manual injec-
tor and Millenium 2.01 software (Waters,
USA). The chromatographic separations
were achieved on a Spherisorb1 C18
(250 mm · 4.6 mm i.d., 5 (m) column
from Waters Corporation. The PDA
analysis was performed on the HPLC
system consisting of a 600 E pump, a 996
photo-diode array (PDA) detector, a 717
autoinjector and a degasser module
(Waters, USA).
Forced Decomposition Studies
Hydrolytic degradation studies were car-
ried out in 0.1 N HCl, water and 0.1 N
NaOH at 85 C as well as at room tem-
perature (r.t.) over 72 h. Oxidative deg-
radation was carried out in 3% H2O2
solution at r. t. over 72 h. For thermal
degradation, the drug was sealed in
borosilicate glass vials and placed in the
hot air oven maintained at 50 C for
31 days. Photolytic studies were carried
out by exposing a thin layer of the solid
drug in a Petri-dish as well as solutions of
the drug in 0.01 N HCl, water and 0.1 N
NaOH to light in the photostability
chamber for 6 weeks during which the
total light exposure equalled 1.2 million
lux h. A parallel set was kept in dark
under similar conditions for 6 weeks. The
drug concentration in all the solution
phase studies was 0.1% w/v. Methanol
(55% v/v) was used for solubilization of
the drug in acidic, neutral and oxidative
media. Samples were withdrawn initially,
subsequently at prefixed time intervals
and stored at 0 C till analysis. Each
sample was neutralised by acid or alkali,
wherever necessary, and diluted ten times
with mobile phase before injection.
Chromatographia 2007, 66, November (No. 9/10)
Separation Method
Detection wavelength for HPLC studies
was selected as 235 nm after recording UV
spectrum (200–400 nm) of the drug and
representative sample from each forced
condition. The mobile phase used initially
was composed of acetonitrile and sodium
dihydrogenphosphate solution (0.02 M).
However, to achieve the optimum resolu-
tion and to render the method LC-MS
compatible, the aqueous phase was re-
placed with ammonium acetate solution.
The chromatographic conditions were
optimized for separation of drug and
degradation products in each forced con-
dition by varying stationary phase,
strength of aqueous phase, pH, proportion
of acetonitrile-aqueous phase and flow
rate using representative samples from
each forced condition. Subsequently a
mixture of a 72 h degradation sample in
water at 85 C, a 6 weeks sample in 0.01 N
HCl exposed to light and 6 weeks sample
in 0.1 N NaOH in the dark was used to fine
tune the chromatographic conditions for
separating gliclazide and all the degrada-
tion products in a single run. The opti-
mized method was used to study the forced
degradation behaviour of gliclazide and
also applied in stability testing of gliclazide
tablets. Appropriate blank was injected
before analysis of the forced samples.
Validation of the Method
The optimized method was validated by
evaluating linearity, precision, accuracy,
specificity, selectivity, ruggedness and
system suitability in accordance with the
ICH guidelines [28].
Linearity was determined by analyz-
ing, in triplicate, standard drug solutions
of concentrations 5, 10, 25, 50, 100, 250,
500 and 1,000 lg mL 1 using 20 lL of
injection volume. For intra-day precision
three drug concentrations (25, 100 and
250 lg mL 1) were analyzed six times on
the same day whereas for inter-day pre-
cision the same drug concentrations were
analyzed on three different days. Accu-
racy was evaluated by fortifying the
mixture of degradation samples (see
‘‘Separation Method’’) with three known
drug concentrations and calculating the
percent recovery from differences be-
tween the peak areas obtained for forti-
fied and unfortified solutions. Specificity
of the method was established through
the study of the resolution factor (Rs) of
Full Short Communication
the drug peak from the nearest resolving
peak. Overall selectivity was established
through determination of purity and Rs
of each peak using a PDA detector.
Ruggedness of the method was estab-
lished through separation studies on the
mixture of degradation samples by dif-
ferent persons on the same chromato-
graphic system as well as on a different
chromatographic system in a different
laboratory on a different day by another
analyst. Various system suitability
parameters were also evaluated on the
mixture sample on six different days by
using freshly prepared mobile phase each
time.

Stability Testing of Gliclazide

Tablets

The packed (blister strip) as well as loose

(removed from the blister pack) gliclazide
tablets placed in a Petri-dish were sub-
jected to accelerated conditions of 40 C/
75% RH in the photostability chamber
for 15 days so that the total light expo-
sure equaled 1.2 million lux. A parallel set
of the packed and loose tablets was kept
in the dark under similar conditions for
15 days. The stability samples were ana-
lyzed by the validated method to quantify
gliclazide and to study the effect of
accelerated conditions on gliclazide in
tablets. Both the loose and packed tablets
from each condition (dark and light) were
processed as follows. Ten tablets were
weighed, powdered and a quantity of the
powder equivalent to 100 mg of gliclazide
was transferred into a 100 mL measuring
flask. About 50 mL of acetonitrile were
added and sonicated for 5 min. The
contents were brought to r.t., volume was
made up with acetonitrile. 5 mL of the
resulting turbid solution was diluted with
mobile phase up to 50 mL in a measuring
flask. Finally, the solution was filtered
through a 0.45 micro membrane and Fig. 2. HPLC chromatograms showing resolution of gliclazide and all the degradation products
(I–VII) in a mixture of forced degradation samples in a single run (a), degradation of gliclazide in
analyzed. 3% H2O2 at r. t. after 72 h (b), in 0.1 N HCl at r. t. after 10 h (c), in water at 85 C after 6 h (d), in
0.1 N NaOH at 85 C after 72 h (e), in 0.01 N HCl (f), water (g) and 0.1 N NaOH (h) exposed to
light and of the packed tablets exposed to light (i)
Results and Discussion

Development and
Optimization of the
Stability-Indicating Method
Initially, the forced degradation samples
were analyzed by using a mobile phase
composed of 65% sodium dihydrogen-
phosphate buffer (0.02 M; pH 3.0) and
35% acetonitrile flowing at a rate of
0.8 mL min 1 on a C18 column employ-
ing 235 nm as detection wavelength. The
method resolved the drug and degrada-
tion products in neutral, acidic and oxi-
dative media, but a cluster of peaks was
observed in alkaline medium. To separate
the degradation products in alkaline
medium and also to characterize the
degradation products by mass spectrom-
etry, an LC-MS compatible method was
targeted. The phosphate buffer was re-
placed with ammonium acetate (0.025 M;
pH 3.0) and all other variables were kept
the same. However, degradation products
in alkaline condition still remained unre-

Full Short Communication Chromatographia 2007, 66, November (No. 9/10) 753
Table 1. Relative retention time (RRT), peak purity data and system suitability parameters of gliclazide and its degradation products

Parameter Peak

VII I II III IV V VI Gliclazide

RRT 0.26 0.30 0.39 0.45 0.52 0.57 0.90 1.00

Purity angle 0.612 0.548 0.693 0.786 0.514 0.691 1.154 0.561
Purity threshold 1.036 0.825 0.874 1.100 0.664 0.839 1.824 0.691
Asymmetry (As) 1.17 1.23 1.20 1.03 1.59 1.05 1.03 1.19
Tailing (T) 1.07 1.13 1.10 1.00 1.33 0.99 1.00 1.09
Retention factor (k) 0.79 1.19 1.78 2.23 2.64 3.00 5.37 6.09
Selectivity (a)a 1.50 1.49 1.25 1.91 1.36 1.79 1.16
Resolution (Rs)a 2.92 3.54 2.92 2.40 1.92 10.12 2.44
Theoretical plates (N) 5,788 2,636 2,698 4,862 1,947 3,886 7,514 4,584

a
with respect to the succeeding peak

Table 2. Intra-day and inter-day precision and accuracy of the method

Precision studies Accuracy studies

Actual conc. Measured concentration Conc. (lg mL 1) Calculated conc. Recovery (%)
(lg mL 1) (lg mL 1) Mean ± SD; %RSD (lg mL 1) Mean ± SD;
%RSD (n = 3)
Intra-day (n = 6) Inter-day (n = 3)

25 28.04 ± 0.20; 0.73 25.27 ± 0.49; 1.92 25 24.95 ± 0.46; 1.84 99.81
100 102.35 ± 0.81; 0.79 97.43 ± 1.81; 1.86 50 50.48 ± 0.66; 1.31 100.97
250 254.22 ± 1.87; 0.74 247.35 ± 2.27; 0.92 125 125.15 ± 1.24; 0.99 100.12

solved. The change in stationary phase
from C18 to C8 also did not yield encour-
aging results. However, a gradual decrease
in flow rate of the mobile phase resulted in
better separation of the products. Even-
tually, mobile phase composed of 60%
ammonium acetate buffer (0.025 M; pH
3.0) and 40% acetonitrile flowing at a rate
of 0.25 mL min 1 over a C18 column
could optimally resolve the drug and all
the degradation products in the mixture
sample in a single run (Fig. 2a). The rel-
ative retention time (RRT) of each peak
with respect to gliclazide is summarized in
Table 1.
Degradation Behaviour
The drug degraded to I and III in oxi-
dative medium at r.t. (Fig. 2b). The deg-
radation behaviour in 0.1 N HCl at r.t.
(Fig. 2c) and in water at 85 C (Fig. 2d)
was similar where I and III were the
major degradation products where as II
and IV were the minor ones. In addition
to I, III and IV, two new products, V and
VI, were also formed in 0.1 N NaOH at
85 C (Fig. 2e) suggesting that the deg-
radation behaviour in alkali was different
from that in acid and water. No degra-
dation was seen in solid drug at 50 C for
31 days and also upon exposure to light
intensity of 1.2 million lux. Drug solu-
tions in water and 0.1 N NaOH under-
went significant degradation in light
(Fig. 2g–h, respectively) with degradation
behaviour similar to that in the dark, but
a new small peak VII was formed in
0.01 N HCl in the light (Fig. 2f). The
product II was the major peak in acidic
and neutral media under photolytic con-
ditions in contrast to the hydrolytic con-
ditions indicating that formation of II
was facilitated at a temperature of 40 C.
Further, in alkaline medium under light
condition, peak area of V increased with
concomitant decrease in VI and disap-
pearance of III. This suggested that VI
and III undergo probably a light cata-
lyzed intermolecular reaction to produce
V which could also be produced by direct
alkaline hydrolysis of the drug. In general
the rate of degradation was accelerated
by light in solution state.
Validation of the Developed
Stability-Indicating Method
The method was strictly linear in the
concentration range of 5–500 lg mL 1.
The mean (±%RSD) values of slope,
intercept and correlation coefficient were
35568 (±3.51), 49687 (±2.81) and 0.9998
(±0.04), respectively. The intra-day and
inter-day precision were <1% and
<2%, respectively (Table 2) confirming
the method to be sufficiently precise.
Excellent recoveries (99.81–100.97%)
were achieved at each added concentra-
tion (Table 2) indicating the method to
be accurate. The method was sufficiently
specific to the drug and selective to the
individual degradation product as indi-
cated by purity angle and threshold
(Table 1). The drug and all the degrada-
tion products resolved from each other
with an Rs of >1.90 (Fig. 2a) during
ruggedness studies indicating that opti-
mum resolution was always achieved
under the tested conditions. The various
system suitability parameters determined
on six different days (Table 1) indicated
that the method is suitable for the pur-
pose. The Rs particularly for the separa-
tion of most closely placed peaks (IV and
V) was always >1.90.
Stability Testing
of the Gliclazide Tablets
No decrease in drug content in loose and
packed tablets kept in the dark at 40 C/
75% RH was observed in comparison to
the control tablets. Product VII was
formed in trace amounts in packed tab-
lets (Fig. 2i) and its level was increased

754 Chromatographia 2007, 66, November (No. 9/10) Full Short Communication
insignificantly in loose tablets upon
exposure to light. However it was <0.1%
of the drug peak and no other peak was
formed in both the packed and loose
tablets.
Conclusion
The forced degradation study on gliclaz-
ide has been carried out under ICH pre-
scribed conditions and an isocratic
stability-indicating HPLC-UV method is
developed. The validation experiments
have proved the method to be linear,
precise, accurate, specific to drug and
selective to each degradation product. In
total, seven degradation products (I–VII)
are formed under different forced condi-
tions and each peak is optimally resolved
from the nearest peak with Rs of at least
1.90. The drug has been found susceptible
to hydrolytic and photolytic degradation
at elevated temperature. The rate of
degradation is accelerated by light and a
photodegradation product is formed in
acidic medium. The method has been
applied in stability testing of the com-
mercially available gliclazide tablets. In
comparison to methods available in the
literature, this method can be applied
to quantify gliclazide as well as its indi-
vidual degradation products during the
stability studies. Also, the chromato-
graphic conditions being compatible with
LC-MS instrument requirements will be
of use to characterize the degradation
products.
Full Short Communication
Acknowledgments
The authors are thankful to Prof. Saranjit
Singh, Head of Department of Pharma-
ceutical Analysis, National Institute of
Pharmaceutical Education and Research,
SAS Nagar, India for providing the
facility of photostability chamber and
PDA analysis for the study. The authors
are also thankful to Punjabi University,
Patiala, India for providing financial
assistance in carrying out the project and
to M/S Panacea Biotec Ltd, Lalru, India
for providing gliclazide as a gift sample.
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