ISSN 10619348, Journal of Analytical Chemistry, 2015, Vol. 70, No. 6, pp. 738–743. © Pleiades Publishing, Ltd., 2015.

ARTICLES

Development and Validation of a StabilityIndicating Reversed Phase

HPLC Method for the Quality Control of Zolpidem
in Bulk and Tablet Dosage Forms1
Saeid Yaripoura, Saeed Nezami Rashida, Hesam Alibakhshia, and Ali Mohammadia, b, c , *
a
Department of Drug and Food Control, Faculty of Pharmacy,
Tehran University of Medical Sciences P.O.Box: 141556451 Tehran, Iran
bPharmaceutical Quality Assurance Research Center, Faculty of Pharmacy,
Tehran University of Medical Sciences Tehran, Iran
cNanotechnology Research Centre, Faculty of Pharmacy, Tehran University of Medical Sciences Tehran, Iran

*email: alimohammadi@tums.ac.ir
Received September 28, 2013; in final form, September 29, 2014

Abstract—An isocratic stabilityindicating reversed phase liquid chromatography (RPHPLC) method for
quantitative determination of Zolpidem in the presence of its stressed degradation products in bulk and com
mercial tablets was developed and validated. Forced degradation studies were carried out on bulk samples and
tablet dosage forms of Zolpidem using acid, base, H2O2, heat, and UV light as described by International
Conference on Harmonization (ICH) for stress conditions to demonstrate the stability indicating power of
the method. A Perfectsil® Target ODS column (3–5 µm, 125 × 4 mm) was used for chromatographic separa
tion. The mobile phase consisted of methanol50 mM ammonium acetate buffer (pH 3.7, 40 : 60, v/v). The
flow rate was 0.7 mL/min and UV detection was optimized at 254 nm. The present method linearity for Zolp
idem was investigated in the range of 1–20 µg/mL (r = 0.9998). The limits of detection (LOD) and quantifi
cation (LOQ) were 400 and 1000 ng/mL respectively. The method selectivity was evaluated by peak purity test
using a diode array detector. There was no interference with detection of Zolpidem and its stressed degrada
tion products.

Keywords: Zolpidem, stabilityindicating, forced degradation, HPLC

DOI: 10.1134/S1061934815060143

1 Zolpidem (Scheme) is chemically described as hypnotic in the shortterm management of insomnia

N,N,6trimethyl2(4methylphenyl)imidazo[1,2a]py with rapid onset and short duration of action. The
ridine3acetamide [1]. usual oral dose is 10 mg taken immediately before
retiring. In elderly or impotent patients, treatment
H3C CH3 should be limited to a dose of 5 mg at night [2–4].
N
CH3 The literature review reveals that several analytical
O methods were reported for determination of Zolpidem
in biological samples including gas chromatography
N [5–8], liquid chromatography [9–20], and capillary
H3C
N electrophoresis [21]. There are a few published analyt
ical methods used for determination of Zolpidem in
Chemical structure of Zolpidem. bulk and pharmaceutical dosage forms such as
Zolpidem is a nonbenzodiazepine sedativehyp HPTLC [22], differential pulse voltammetry [23],
notic drug that became available in the United States simple spectrophotometry [24] and HPLC [25, 26]. In
in 1993 after 5 years of being used in Europe. However, order to apply of an analytical method for quality con
the action of zolpidem is due to the selective agonist of trol analysis in pharmaceutical industries, it is pre
the omega1 receptor at the CNS gamma amino ferred that any analytical method should be stability
butyric acid/chloride channel complex; it is not a ben indicating [27–30]. There is no information about the
zodiazepine, but an imidazopyridine. In the experi stabilityindicating nature of the analytical method
mental process only weak anticonvulsant effects on that was defined as Zolpidem monograph in the
animals were observed from Zolpidem. It is used as a United States Pharmacopeia [31]. Only one of the
published HPLC methods [26] was apparently intro
1 The article is published in the original.
duced as stabilityindicating. In that study, the selec

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 DEVELOPMENT AND VALIDATION OF A STABILITYINDICATING REVERSED PHASE
tivity of the method (peak purity test) was not evalu
ated and the method was assessed by simple UV detec
tion which is not suitable for investigation of peak
purity. Also in that study oxidative (using H2O2) and
thermal stress studies were performed at high temper
atures (>70°C) which were not recommended by ICH:
the O–O bond in H2O2 is weak, and using the temper
atures more than 40°C leads to cleaving that bond
quickly with forming the hydroxyl radicals which are
the aggressive oxidative reagents and usually were not
formed under longterm storage conditions. Under
the high temperature, drug degradation kinetics does
not follow the Arrhenius equation. In conclusion,
regarding our knowledge in pharmaceutical stress test
ing studies, the temperature increment (> 70°C) may
change the mechanisms and the nature of degradation
products which were not formed in longterm stability
studies [27, 32]. So it is not reasonably assessment cri
teria to predict the drug shelf life in quality control
procedures.
The purpose of our study was to develop and vali
date a stabilityindicating RPHPLC method for
determination of Zolpidem in the presence of its deg
radation products in bulk and tablet dosage forms
according to the ICH guidelines, the result which was
not obtained in previously developed methods. Our
proposed method is rapid, precise, accurate, selective,
economical, and has been successfully applied for
determination and quality control of Zolpidem in bulk
samples and tablet dosage forms.
EXPERIMENTAL
Materials and reagents. Zolpidem tartrate was
kindly supplied by Farmak (Czech Republic). Stil
nox® 10 mg tablets were obtained from Sanofi Aventis
(Sanofi Winthrop Industrie, France). Methanol,
ammonium acetate, acetic acid (glacial), hydrochloric
acid, sodium hydroxide and hydrogen peroxide were
purchased from Merck (Darmstadt, Germany). All
reagents were analytical grade except methanol which
was HPLC grade. All solutions prepared by HPLC
grade water that was obtained after purification by a
MilliQ® system (Millipore, Milford, MA, USA).
Chromatographic conditions. The modular HPLC
system consisted of Knauer HPLC Pump K1001, a
UV Detector K2600, a Knauer injection system and
a Knauer solvent degasser (Berlin, Germany). The
ChromGate® Chromatography Data System Version
3.1.7 (Berlin, Germany) was applied to record and
analyze the chromatographic data. A Perfectsil® Tar
get ODS column (3–5 μm, 125 × 4 mm, MZ Analy
sentechnik, Mainz, Germany) was used for chromato
graphic separation. The mobile phase was consisted of
methanol−50 mM ammonium acetate buffer (40 : 60,
v/v, pH 3.7). The flow rate was 0.7 mL/min and UV
detection was optimized at 254 nm. The column tem
perature was 25 ± 2°C and volume of every injection
was 50 μL. The buffer was filtered through 0.45 μm
JOURNAL OF ANALYTICAL CHEMISTRY Vol. 70
739
PTFE membrane filter before using. For peak purity
evaluation, all unstressed and stressed samples were
injected to Alliance Waters 2998 HPLC system with
Waters diode array detector (2998 PDA, Waters, USA)
under the same conditions mentioned above. The
results were analyzed by Empower PDA Software
1154.
Preparation of stock and standard solutions. A stock
solution of Zolpidem (200 μg/mL) was prepared in
methanol. This solution was protected from light and
stored at 4°C for 3 weeks without any decomposition.
Aliquots of the standard stock solution of Zolpidem
were transferred into 10 mL Agrade volumetric flasks
using Agrade bulb pipettes, and the solutions were
made up to get volume by mobile phase for obtaining
solutions with final concentrations of 1, 2, 5, 10 and
20 μg/mL.
Preparation of tablets. Twenty Stilnox® 10 mg tab
lets were weighed and finely powdered. A quantity of
powder equal to one tablet containing 10 mg of Zolpi
dem was transferred in a volumetric flask and HPLC
grade methanol was added. Then the solution was son
icated for 10 min, adjusted to the volume and again
diluted by mobile phase. Finally the solution with final
concentration of 5 μg/mL was obtained. The solution
was filtered and injected to HPLC.
Stressed degradation studies of Zolpidem active
pharmaceutical ingredient (API) and tablet powder. To
prove the stabilityindicating nature of the proposed
method, Stilnox® tablets and pure API of Zolpidem
were stressed under various conditions [27–30]. In
order to perform forced degradation studies, API and
tablet dosage form of Zolpidem were dissolved in dis
tilled water and diluted with aqueous H2O2, distilled
water, aqueous HCl, and aqueous NaOH, to obtain
concentrations of 200 μg/mL. After the degradation,
all solutions were diluted by mobile phase and filtered
to obtain concentrations of 20 μg/mL for injection
into HPLC.
Oxidative degradation studies. In order to perform
oxidative degradation studies, solutions were prepared
in water and 30% H2O2 (10 : 90, v/v) at room temper
ature (25 ± 2°C). The resultant solutions were ana
lyzed 200 min (time of degradation) after preparation.
Acid degradation studies. Solutions for acid degra
dation studies were prepared in water and 1 M HCl
(10 : 90, v/v) and heated at 70°C. After 120 min deg
radation, the resultant solutions were analyzed by
HPLC.
Base degradation studies. Solutions for base degra
dation studies were prepared in water and 1 M NaOH
(10 : 90, v/v) and heated at 70°C. After 90 min degra
dation, the resultant solutions were analyzed by
HPLC.
Thermal degradation studies. Zolpidem API and
tablet dosage form were exposed to heat (70°C) in a
convection oven. After 5 days degradation, a solution
No. 6 2015