Application of spectrophotometric, densitometric, and HPLC techniques as stability indicating methods for determination of Zaleplon in pharmaceutical preparations

Article abstract of DOI:10.1016/j.saa.2007.01.024

Spectrophotometric, spectrodensitometric and HPLC are stability indicating methods described for determination of Zaleplon in pure and dosage forms. As Zaleplon is easily degradable, the proposed techniques in this manuscript are adopted for its determination in presence of its alkaline degradation product, namely N-[4-(3-cyano-pyrazolo[1,5a]pyridin-7-yl)-phenyl]-N-ethyl-acetamide. These approaches are successfully applied to quantify Zaleplon using the information included in the absorption spectra of appropriate solutions. The second derivative (D₂) spectrophotometric method, allows determination of Zaleplon without interference of its degradate at 235.2 nm using 0.01N HCl as a solvent with obedience to Beer's law over a concentration range of 1-10 μg ml^-1 with mean percentage recovery 100.24 ± 0.86%. The first derivative of the ratio spectra (¹DD) based on the simultaneous use of (¹DD) and measurement at 241.8 nm using the same solvent and over the same concentration range as (D₂) spectrophotometric method, with mean percentage recovery 99.9 ± 1.07%. The spectrodensitometric analysis allows the separation and quantitation of Zaleplon from its degradate on silica gel plates using chloroform:acetone:ammonia solution (9:1:0.2 by volume) as a mobile phase. This method depends on quantitave densitometric evaluation of thin layer chromatogram of Zaleplon at 338 nm over a concentration range of 0.2-1 μg band^-1, with mean percentage recovery 99.73 ± 1.35. Also a reversed-phase liquid chromatographic method using 5-C8 (22 cm × 4.6 mm i.d. 5 μm particle size) column was described and validated for quantitation of Zaleplon using acetonitrile:deionised water (35:65, v/v) as a mobile phase using Paracetamol as internal standard and a flow rate of 1.5 ml min^-1 with UV detection of the effluent at 232 nm at ambient temperature over a concentration range of 2-20 μg ml^-1 with mean percentage recovery 100.19 ± 1.15%. The insignificance difference of the proposed methods results with those of the reference one proved their accuracy and precision.

Full text of DOI:10.1016/j.saa.2007.01.024

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Spectrochimica Acta Part A 68 (2007) 1220–1230
Application of spectrophotometric, densitometric, and HPLC
techniques as stability indicating methods for determination
of Zaleplon in pharmaceutical preparations
Fadia H. Metwally∗, M. Abdelkawy, Nada S. Abdelwahab
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., 11562 Cairo, Egypt
Received 7 October 2006; received in revised form 14 January 2007; accepted 24 January 2007
Abstract
Spectrophotometric, spectrodensitometric and HPLC are stability indicating methods described for determination of Zaleplon in pure and dosage
forms.
As Zaleplon is easily degradable, the proposed techniques in this manuscript are adopted for its determination in presence of its alkaline
degradation product, namely N-[4-(3-cyano-pyrazolo[1,5a]pyridin-7-yl)-phenyl]-N-ethyl-acetamide. These approaches are successfully applied to
quantify Zaleplon using the information included in the absorption spectra of appropriate solutions.
The second derivative (D2) spectrophotometric method, allows determination of Zaleplon without interference of its degradate at 235.2 nm using
0.01N HCl as a solvent with obedience to Beer’s law over a concentration range of 1–10 ␮g ml−1 with mean percentage recovery 100.24 0.86%.
The first derivative of the ratio spectra (1DD) based on the simultaneous use of (1DD) and measurement at 241.8 nm using the same solvent and
over the same concentration range as (D2) spectrophotometric method, with mean percentage recovery 99.9 1.07%.
The spectrodensitometric analysis allows the separation and quantitation of Zaleplon from its degradate on silica gel plates using chloro-
form:acetone:ammonia solution (9:1:0.2 by volume) as a mobile phase. This method depends on quantitave densitometric evaluation of thin layer
chromatogram of Zaleplon at 338 nm over a concentration range of 0.2–1 ␮g band−1, with mean percentage recovery 99.73 1.35.
Also a reversed-phase liquid chromatographic method using 5-C8 (22 cm × 4.6 mm i.d. 5 ␮m particle size) column was described and validated
for quantitation of Zaleplon using acetonitrile:deionised water (35:65, v/v) as a mobile phase using Paracetamol as internal standard and a flow
rate of 1.5 ml min−1 with UV detection of the effluent at 232 nm at ambient temperature over a concentration range of 2–20 ␮g ml−1 with mean
percentage recovery 100.19 1.15%.
The insignificance difference of the proposed methods results with those of the reference one proved their accuracy and precision.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Zaleplon; Derivative spectrophotometry; Derivative ratio spectrodensitometry; HPLC
1. Introduction
ular weight and molecular formula of Zaleplon are illustrated in
Fig. 1.
Zaleplon is N-[3-(3-cyanopyrazolo[1,5-a]pyrimidin-7-yl)
phenyl]-N-ethyl acetamide [1]. It is a pyrazolopyrimidin deriva-
tive. Although not structurally related to the benzodiazepines,
Zaleplon acts through binding to the ␥-amino butyric acid
(GABAA)-benzodiazepinereceptorcomplexproducingsedative
and hypnotic effects similar to those of benzodiazepines [2–4].
It is reported to have relative selectivity for the ꢀ1-subtype of
benzodiazepinesbindingsite[5]. Thechemicalstructure, molec-
The literature survey reveals several methods for determi-
nation of Zaleplon in plasma, namely, liquid chromatography–
electro spray ionisation–mass spectrometry assay, high-
performance liquid chromatography, chemical ionisation–mass
spectrometry [6–8], and RP-HPLC with fluorescence detection
[9]. A spectrofluorimetric technique is used for its determi-
nation in micellar medium [10]. Zaleplon was found among
drugs screened in hair and oral fluids using LC–MS [11,12].
It was quantitatively screened in blood as silylated derivative by
gas chromatography–selected ion monitoring mass spectrom-
etry and gas chromatography electron capture detection [13].
A fast gas chromatography–negative-ion chemical ionization
∗
Corresponding author. Tel.: +20 2 417 8691; fax: +20 2 362 4105.
E-mail address: fadiahm@yahoo.com (F.H. Metwally).
1386-1425/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2007.01.024

F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
1221
The following requirements are taken into consideration:
- Slite dimensions = 6.00 × 0.445, Micro.
- Scanning speed = 20 mm s−1
.
- Data resolution = 100 ␮m step−1
.
4- Sample applicator for TLC linomat IV with 100 ␮l syringe
(Camage, Muttenz, Switzerland).
5- TLC plates (20 cm × 20 cm) coated with silica gel 60 F254
(Merck KgaA, Darmstad, Germany).
6- The HPLC system consisted of a Perkin Elmer system
equipped with series 200 auto sampler, series 200 lc pump,
series 200 UV/vis detector. The stationary phase was 5-C8
(22 cm × 4.6 mm i.d. 5 ␮m particle size) column using ace-
tonitrile:deionised water (35:65, v/v) as a mobile phase.
2.2. Materials
2.2.1. Authentic samples
1- Zaleplon (Batch No. 0550407) was kindly supplied by The
Egyptian Co. for Pharmaceutical and Chemical Industries.
S.A.E (EPCI), Industrial Zone, Bayad El-Arab, Beni Suef,
Egypt. Its purity was reported to be 98.5% according to the
company analysis certificate.
2- Paracetamol (Batch No. 34055) was kindly supplied by
Memphis Co. for Pharm and Chemical Ind., Cairo, Egypt. Its
purity reported to be 99% according to the company analysis
certificate.
Fig. 1. The chemical structure, molecular weight and molecular formula of
Zaleplon followed by the degradation pattern of Zaleplon in 0.05N NaOH.
mass spectrometry with micro scale volume samples prepara-
tion method was described for its determination in blood [14].
It was determined in capsules by voltammetric method [15]. A
capillary electrophoresis with laser-induced fluorescence detec-
tion and liquid chromatography–mass spectrometry techniques
were adopted for separation and identification of its metabolites
in human urine [16].
The above literature revealed that up to the present time noth-
ing has been published concerning the proposed methods for
determination of Zaleplon in presence of its alkaline degradation
product.
2.2.2. Dosage form
1- Siesta® capsules(BatchNo. 50507), labeledtocontain10 mg
Zaleplon is from SIGMA For Al Andalous Medical Com-
pany, Cairo, Egypt.
2- Zalocid® capsules (Batch No. 044060609/3), labeled to
contain 10 mg Zaleplon is from The Egyptian Co. for Phar-
maceutical and Chemical Industries. S.A.E. (EPCI).
2.2.3. Chemicals and solvents
The proposed procedures were successfully applied to the
routine and quality control analysis of Zaleplon in its pharma-
ceutical preparation.
1- Chloroform, hydrochloric acid, acetone, ammonia solution,
NaOH and methanol all are from (El NASR Pharmaceutical
Chemicals Co., Abu-Zabaal, Cairo, Egypt).
2- Acetonitrile HPLC grade (SDS, France).
3- Deionised water (SEDICO pharmaceuticals Co., Cairo,
Egypt).
2. Experimental
N.B.: All the chemicals were of analytical grade. The
solvents used for the spectrophotometric and spectrodensito-
metric methods were of spectrophotometric grade and those
used for HPLC were of HPLC grade.
2.1. Apparatus
1- Shimadzu UV–vis 1601 PC spectrophotometer with 1 cm
quartez cuvettes for spectrophotometric determinations
(Kyoto, Japan).
The following requirements are taken into consideration:
- Scan speed: fast.
2.2.4. Degraded samples
2.2.4.1. Preparation of pure degraded sample. 0.5 g Zaleplon
powder was transferred to 250 ml stoppered flask, water refluxed
with 100 ml 0.05N NaOH with magnetic stirring for 3 h. The
solution was cooled, the collected precipitate contains a mix-
ture of Zaleplon and its degradate, from which the degradate
was separated in pure form by preparative normal phase column
chromatographyusingsilicagelasastationaryphaseandchloro-
- ꢁλ: 4 (for both D2 and 1DD methods).
- Scaling factor: 100 (for D2 method) and 10 (for 1DD
method).
2- UV lamp with short wavelength 254 nm (USA).
3- TLC Scanner
Switzerland).
3
densitometer (Camage, Muttenz,

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F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
form as a mobile phase with gradient increase in polarity by 1%
acetone (using 2 ml ammonia as constant volume) each 100 ml
addition. Fractions were collected each 10 ml and the degra-
date was identified by TLC using chloroform:acetone:ammonia
(9:1:0.2, v/v/v) as mobile phase. Similar fractions were col-
lected together and allowed to dry at room temperature until
constant weight. The dry powder was identified by IR and the
mass spectrometry.
4.1.2. Assay of laboratory prepared mixtures for D2 and
1DD spectrophotometric methods
The D2 and the ratio spectra of first derivative spec-
trophotometric curves of different laboratory prepared mixtures
containing different ratios of Zaleplon and its degradate were
recorded. The peak amplitudes at 235.2 and 241.8 nm were
1
recorded for D2 and DD, respectively. The concentration of
Zaleplon was calculated from the corresponding regression
equations.
3. Standard preparations
4.2. Spectrodensitometric method
3.1. Stock solutions
4.2.1. Linearity and construction of calibration curve
Accurately measured aliquots equivalent to (0.2–1 ␮g) of
Zaleplon working solution (100 ␮g ml−1) were applied to a thin
layer chromatographic plate (20 × 20) as a band using the Cam-
age TLC sampler. Leave a space of 1 cm between each band and
1.5 cm from the bottom edge of the plate. Develop the plate in
a chromatographic tank previously saturated for an hour with
the developing mobile phase, chloroform:acetone:ammonia
(9:1:0.2, v/v/v) by ascending chromatography at room temper-
ature. Zaleplon bands were scanned at 338 nm. The calibration
curve was constructed by plotting the peak height/1000 versus
the corresponding concentration and the regression equation was
developed.
3.1.1. For D2, 1DD spectrophotometric and
spectrodensitometric methods
Stock solutions for each of Zaleplon and the degradate are
prepared by weighing accurately 0.1 g of pure powder of each,
transfer into two separate 100-ml volumetric flasks, add 75 ml
methanol, shake well then complete to the mark with the same
solvent to get 1000 ␮g ml−1 stock solution for each.
3.1.2. For HPLC method
For Zaleplon, the degradate and internal standard (Paraceta-
mol) were prepared as above but using acetonitrile:deionised
water (35:65, v/v) as a solvent.
3.2. Working solutions
4.2.2. Analysis of laboratory prepared mixtures
The peak heights of different laboratory prepared mixtures
containing different ratios of Zaleplon and the degradate were
measured and the concentration of Zaleplon in each mixture was
obtained by applying in the corresponding regression equation.
3.2.1. For D2 and 1DD spectrophotometric methods
Ten millilitres of each of Zaleplon and its degradate were sep-
arately transferred into two separate 100-ml volumetric flasks,
completed to the mark with 0.01N HCl to get 100 ␮g ml−1
working solutions.
4.3. HPLC method
3.2.2. For spectrodensitometric method
4.3.1. Linearity and construction of calibration curve
For each of Zaleplon and the degradate working solutions
proceed as above but using methanol as a solvent.
Aliquots equivalent to (2–20 ␮g) of Zaleplon working solu-
tion (100 ␮g ml−1) were separately transferred into a series of
10-ml volumetric flasks, 1.5 ml of Paracetamol working solu-
tion (100 ␮g ml−1) was mixed with each one then completed to
the volume with acetonitrile:deionised water (36:65, v/v). Trip-
licate 20 ␮g ml−1 injection were made for each concentration.
Chromatograms were recorded under the following instrumental
parameters: flow rate was 1.5 ml min−1 at ambient temperature
and the effluent was monitored at 232 nm. The separation was
done on C8 column using acetonitrile:deionised water (36:65,
v/v) as a mobile phase. The peak areas of Zaleplon and Parac-
etamol were recorded and the peak area ratio was calculated for
each concentration. The calibration curve relating the obtained
peak area ratio to corresponding concentration was constructed
and the regression equation was then computed.
3.2.3. For HPLC method
For Zaleplon, Paracetamol and the degradate proceed as
above but using acetonitrile:water (35:65, v/v) as a solvent.
4. Procedures
4.1. D2 and 1DD spectrophotometric methods
4.1.1. Linearity and construction of calibration curve
The D2 absorption spectra of Zaleplon in the range of
(1–10 ␮g ml−1) were recorded using 0.01N HCl as a blank (for
D2 spectrophotometric method). The absorption spectra of Zale-
plon in the same range were divided by the absorption spectrum
of 6 ␮g ml−1 of the degradate (the divisor). The obtained ratio
spectra were differentiated with respect to wavelength (for 1DD
method). The amplitude values at 235.2 and 241.8 nm were mea-
sured for both D2 and 1DD, respectively. The calibration graphs
were then plotted, and regression equations were computed.
4.3.2. Analysis of laboratory prepared mixtures
The chromatographic conditions used under linearity and
construction of calibration curve were applied for different
laboratory prepared mixtures containing different ratios of Zale-
plon and the degradate. The peak area ratio of Zaleplon and

F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
1223
Paracetamol was calculated for each mixture, Zaleplon con-
centration was calculated by substituting in the regression
equation.
which may not fall in correspondence of peaks of the deriva-
tive spectrum. This may be dangerous when the slope of the
spectrum is very high with consequent loss of accuracy and pre-
cession [19]. In the present case, the above circumstances did
not occur.
4.4. Application to pharmaceutical formulation
The spectrodensitometric and HPLC methods were evaluated
to improve the selectivity and sensitivity of Zaleplon analysis in
presence of its degradation product.
Few procedures were found in the literature for determination
of Zaleplon in absence of its degradation product. The proposed
methods are devoted for the analysis of Zaleplon alone or in
presence of its degradation product.
The content of 20 capsules of each of Siesta® capsules and
Zalocid® capsules were separately emptied and mixed well, an
amount of the powder of each equivalent to 0.1 g of Zaleplon
was accurately weighed into 100-ml volumetric flask, 75 ml of
appropriate solvent added, stirred magnetically forabout 30 min,
cooled well then completed to the volume to get 1000 ␮g ml−1
stock solution then filtered. Ten millilitres of the stock solu-
tion was accurately transferred into 100-ml volumetric flask,
completed to the volume to get 100 ␮g ml−1 working solution,
then the procedure detailed under linearity and construction of
calibration curve for each method was followed.
5.1. D2 spectrophotometric method
The zero-order absorption spectra of Zaleplon and its degra-
date showed a serious overlapping (Fig. 3), that did not allow the
useofdirectspectrophotometricanalysisofZaleploninpresence
of its degradate.
Derivative spectrophotometry has been applied extensively to
the simultaneous determination of substances with overlapping
spectra, which is frequently made on the basis of zero-crossing
measurements.
When carrying out the standard addition technique, the pow-
der content of the capsules and that of pure Zaleplon were mixed
well together before proceeding in the above mentioned proce-
dure.
5. Results and discussion
Different solvents were tried to resolve this overlapping, e.g.
methanol, absolute ethanol, acetonitrile, ethyl acetate, 0.01N
NaOH and 0.01N HCl. In each of these cases the first, second
and third derivatives were checked. By applying D2 technique
zero crossing point for Zaleplon with the degradate was shown
at 235.2 nm (Fig. 4).
A linear correlation was obtained between the peak amplitude
and the concentration in the range of 1–10 ␮g ml−1 from which
the linear regression equation was computed and found to be:
The high susceptibility of Zaleplon to hydrolysis makes its
analysis in presence of its alkaline degradation product an ana-
lytical task of potential.
Upon degradation with sodium hydroxide, the separated
degradate was confirmed using the IR and mass spectra anal-
ysis. The IR spectrum of the degradate showed that the peak
of carbonyl group present in the parent compound was shifted
from 1651 to 1660 cm−1 as broad peak indicating the presence
of two carbonyl groups in the degradate. Peak of cyanide group
which appeared in the drug at about 2231 cm−1 disappeared
and a new one at 3399 cm−1 appeared indicating the presence
of hydroxyl group. Presence of peak at 2982 cm−1 (of aliphatic
protons of amide) in both drug and degradate indicated that the
amide group was unaffected by the used hydrolysis conditions.
The mass spectra confirm these claims where the base peak for
each compound corresponds to its molecular weight (Fig. 2).
On the other hand, hydrolsis using 0.05N HCl for 30 min was
checked using TLC which showed that Zaleplon degraded com-
pletely giving three spots of minor components in addition to
the major degradate. This caused great difficulty in separating
and identifying these degradation products. The same results
obtained on oxidation with hydrogen peroxide.
Y = 0.0749C + 0.0025, r = 0.9999
where Y is the peak amplitude at 235.2 nm, C the concentration
in ␮g ml−1 and r is the correlation coefficient.
5.1.1. Optimization of D2 spectrophotometric method
To optimize D2 spectrophotometric method it was necessary
to test the influence of different variables. Different smoothing
factors (ꢁλ) values given by the program (2, 4 and 8) were tried
where a smoothing factor = 4 showed a suitable signal to noise
ratio and the spectra showed good resolution. Different scaling
factor values (10, 100, and 1000) were tried where scaling factor
100 was suitable to enlarge the signal of Zaleplon to facilitate
its measurement and to diminish error in reading signal.
This method is valid for determination of Zaleplon in pres-
ence of up to 60% of the degradate without interference with
mean percentage recoveries of 101.29 1.19% (Table 1).
Also the method was applied to assay Zaleplon in its phar-
maceutical formulations, and its validity was further assessed
by applying the standard addition technique (Table 3).
D3 spectrophotometric method can also be used to resolve
spectral overlapping of the absorption spectra of Zaleplon and its
degradate at 226.4 nm (ꢁλ = 8, scaling factor = 100) (Fig. 5), but
it was less selective than using D2 spectrophotometric method
(Table 2).
New methods for simultaneous determination of two or more
compounds in the same sample without previous chemical sep-
aration are always of interest.
Derivative spectrophotometry offers greater selectivity than
does the normal spectrophotometry, as it decrease spectral over-
lap and allows better resolution [17].
The 1DD spectrophotometric method has an advantage that
the measurements are carried out at peaks, hence there is a
potential for greater sensitivity and accuracy [18]. The main
disadvantage of zero crossing method in derivative spectropho-
tometry is the risk of small drifts of the working wavelengths,

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F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
Fig. 2. IR and mass spectra of Zaleplon and its degradation product. (a) IR of Zaleplon, (b) IR of the degradate, (c) mass spectra of Zaleplon, and (d) mass spectra
of the degradate.
5.2. 1DD spectrophotometric method
To solve the problem of overlapping absorption spectra of
Zaleplon and its degradate, Zaleplon can be assayed by divid-
ing the absorption spectra of its different concentrations in
the range (1–10 ␮g ml−1) by the absorption spectrum of the
degradate (6 ␮g ml−1), the obtained ratio spectra were differ-
The proposed technique is based on the use of first
derivative of the ratio spectra for resolving binary mixtures
[17].
Fig. 3. Zero-order spectra of 8 ␮g ml−1 of Zaleplon (—) and 4 ␮g ml−1 of the
Fig. 4. The second derivative spectra of 8 ␮g ml−1 of Zaleplon and 4 ␮g ml−1
degragate (- - -) in 0.01N HCl.
of the degradate (Spectrum indicated by arrow) in 0.01N HCl.

F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
1225
Table 1
Spectrophotometric determination of laboratory prepared mixtures for Zaleplon in presence of its degradation product by the second derivative (D2) at 235.2 nm and
the first derivative of ratio spectra (1DD) at 241.8 nm methods
Mixture no.
Zaleplon %
Taken (␮g ml−1
)
Second derivative (D2 method)
First derivative of ratio spectra (1DD method)
Founda (␮g ml−1
)
Found %
Founda (␮g ml−1
)
Found %
1
2
3
4
5
6
7
8
90
80
70
60
50
40
30
20
5.40
4.80
4.20
3.60
3.00
2.40
1.80
1.20
5.39
4.91
4.28
3.60
3.08
2.43
99.82
102.29
101.91
100.00
102.67
101.25
5.49
4.89
4.26
3.60
3.01
2.41
1.81
1.19
101.76
101.88
101.43
100.00
100.33
100.42
100.56
99.17
Mean S.D.
101.29 1.19
100.69 0.93
a
Average of three experiments.
Fig. 5. The third derivative spectra of 8 ␮g ml−1 of Zaleplon (—) and 4 ␮g ml−1
of the degradate (· · ·) in 0.01N HCl.
Fig. 6. First derivative of ratio spectra of 10 ␮g ml−1 of Zaleplon (—) and
8 ␮g ml−1 of the degradate (- - - ) in 0.01N HCl.
entiated with respect to wavelength (ꢁλ = 4, scaling factor = 10)
(Fig. 6).
DD1 values showed good linearity and accuracy at
λ = 241.8 nm. The linear regression equation was found to be:
a- The divisor concentration: different concentrations of the
degradate were tested, but concentration of (6 ␮g ml−1
)
give the best results recording to sensitivity and
selectivity.
Y = 0.2974C + 0.0248, r = 0.9999
b- Different smoothing factors (ꢁλ) values were tried where a
smoothing factor = 4 showed a suitable signal to noise ratio
and the spectra showed good resolution. Different scaling
factor values were tried where scaling factor 10 was suitable
to enlarge the signal of Zaleplon to facilitate its measurement
and to diminish error in reading signal.
where Y is the peak amplitude at 241.8 nm, C the concentration
in ␮g ml−1 and r is the correlation coefficient.
5.2.1. Optimization of 1DD spectrophotometric method
To optimize this method, it was necessary to test the influence
of variables:
Table 2
Laboratory prepared mixtures for determination of Zaleplon in presence of its degradation product by the second derivative D2, third derivative (D3) and the first
derivative of ratio spectra (1DD) (at different wave lengths) spectrophotometric methods
Zaleplon % in the mixture
Second derivative (D2) at 235.2 nm
Third derivative (D3) at 226.4 nm
First derivative of ratio spectra (1DD)
At 259.6 nm
At 241.8 nm
At 225.4 nm
Found %
90
80
70
60
50
40
30
20
99.82
102.29
101.91
100.00
102.67
101.25
109.44a
100.94
101.10
102.26
102.31
106.10a
99.78
102.27
99.48
101.03
99.87
107.96a
101.76
101.88
101.43
100.00
100.33
100.42
100.56
99.17
100.11
99.71
100.95
99.47
101.77
99.58
100.06
90.92a
Mean S.D.
101.29 1.19
101.65 0.73
100.49 1.16
100.69 0.93
100.24 0.84
a
Using the rejection rule.

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F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
Table 3
Application of standard addition technique to the analysis of Zaleplon in capsules by second derivative (D2) at 235.2 nm and the first derivative of ratio spectra (1DD)
at 241.8 nm
Taken (␮g ml−1
)
Second derivative (D2)
method method
First derivative of ratio
spectra (1DD method)
Pure added
Second derivative (D2)
First derivative of ratio
spectra (1DD)
(␮g ml−1
)
Founda
(␮g ml−1
Found (%)
Founda
(␮g ml−1
Found (%)
Pure foundb
Recovery %
Pure foundb
(␮g ml−1
)
Recovery %
)
)
(␮g ml−1
)
Siesta® capsules (Batch No. 50507)
5.00
2.00
3.00
4.00
5.00
1.98
2.94
4.01
5.09
99.00
98.00
100.25
101.80
1.96
3.02
3.95
5.05
98.00
100.67
98.75
4.86
97.20
4.87
4.83
97.40
101.00
Mean S.D.
99.76 1.64
99.61 1.46
Zalocid® capsules (Batch No. 044060609/3
5.00
2.000
3.000
4.000
5.000
2.02
2.99
3.97
5.08
101.00
99.67
99.25
1.98
3.06
4.00
5.00
99.00
102.00
100.00
100.00
4.80
96.00
96.60
101.60
Mean S.D.
100.38 1.10
100.25 1.26
a
Average of six experiments.
Average of three experiments.
b
Results obtained by the proposed technique showed that the
degradate was achieved by using chloroform:acetone:ammonia
(9:1:0.20, v/v/v).
concentration of Zaleplon can be determined in the presence of
up to 80% of the degradate in the laboratory prepared mixtures
with mean percentage recovery 100.69 0.93% (Table 1).
This method was successfully applied to the determination
of Zaleplon in pharmaceutical formulations and applying the
standard addition technique assessed the validity of this method
(Table 3).
Also different scanning wavelengths were tested, but the best
sensitivity obtained when Zaleplon was scanned at 338 nm.
Results obtained in Table 4 showed that this method is
selective, valid and applicable for determination of Zaleplon in
presence of up to 90% of its degradate with mean percentage
recovery 101.39 1.53%.
1DD can also be determined at 225.6 and 259.6 nm (Fig. 6),
where the 1DD value showed good linearity and accuracy but of
little selectivity than at 241.8 nm (Table 2).
The validity of the method was further assessed by applying
the standard addition technique (Table 5).
5.4. HPLC method
5.3. Spectrodensitometric method
A validated isocratic RP-HPLC method with UV detection
was developed for the quantitation of Zaleplon in presence of
its degradation product.
This technique offers a simple way to quantify directly on
TLC plate by measuring the optical density of the separated
bands. The amounts of compounds are determined by comparing
the peak height of the unknown band to a standard curve from
reference materials chromatographed simultaneously under the
same condition [20].
The method is based on the difference in the Rf values of
Zaleplon (Rf = 0.447) and the degradate (Rf = 0.145) (Fig. 7).
The calibration curve was constructed by plotting the peak
height (1/1000) versus the concentration in the range of
0.2–1 ␮g band−1, Zaleplon concentration was calculated from
the following regression equation:
Y = 0.2174X + 0.0504, r = 0.9996
where Y is the peak height (1/1000), X the concentration in
␮g band−1 and r is the correlation coefficient.
5.3.1. Optimization of spectrodensitometric method
Studying of the optimum parameters for maximum sepa-
ration was carried out by trying different developing systems
with different ratios, but complete separation of Zaleplon and its
Fig. 7. 3D diagram showing an example of separated mixture of Zaleplon (a)
and the degradate (b) in the spectrodensitometric method.

F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
1227
Table 4
that of C18 column, although they have the same sensitivity and
selectivity.
Laboratory prepared mixtures for determination of Zaleplon in presence of its
degradate by spectrodensitometric method at 338 nm
Concerning the mobile phase, different systems were tried
for chromatographic separation of the drug from its degradate by
combining homogenous design and solvent polarity optimiza-
tion. The best resolution was achieved when using a mobile
phase consisting of acetonitrile:water (35:65, v/v).
Also the effect of pH was studied by using buffers of different
pH values. It was found that changing the pH does not affect the
peak shape.
Finally, a satisfactory separation was obtained by using ace-
tonitrile:water (35:65, v/v) as a mobile phase, maintaining the
flow rate of 1.5 ml min−1 with UV detection at 232 nm.
Paracetamol was used as internal standard, where the reten-
tion time of Paracetamol was 1.69 min, the degradate was
2.5 min and Zaleplon was 4.06 min, with no interference
among the three peaks. Typical chromatogram is shown in
Fig. 8.
The efficiency of the proposed method as stability indicating
one was assessed by applying analysis of laboratory prepared
mixtures containing different ratios of intact Zaleplon and its
degradation product, where Zaleplon could be determined in
presence of up to 90% of the degradation products without inter-
ference, with mean percentage recoveries of 101.47 0.83%
(Table 6).
This method was applied to assay Zaleplon in its in dosage
forms. The validity of the method was further assessed by apply-
ing the standard addition technique (Table 7).
Mixture no.
Zaleplon % Taken
(␮g band−1
Founda
Found %
)
(␮g band−1
)
1
2
3
4
5
6
7
8
9
90
80
70
60
50
40
30
20
10
0.54
0.55
0.32
0.69
0.49
0.62
0.57
0.43
0.41
0.20
101.85
100.00
98.57
102.08
103.33
101.79
102.38
102.5
0.32
0.70
0.48
0.60
0.56
0.42
0.40
0.20
100.00
Mean S.D.
101.39 1.53
a
Average of three experiments.
It depends on the chromatographic separation of the drug and
itsdegradateusing5-C8(22 cm × 4.6 mmi.d. 5 ␮mparticlesize)
column and acetonitrile:water (35:65, v/v) as a mobile phase
with UV detection at 232 nm. This method has the advantage
of using an internal standard (Paracetamol), which compensates
for any error that may occur due to baseline drift or fluctuations
in readings of UV detectors.
A linear correlation was obtained between the peak area ratio
and Zaleplon concentration in the range of 2–20 ␮g ml−1. The
regression equation was calculated and found to be:
Y = 0.162X − 0.0032, r = 0.9998
where Y is the peak area ratio (of Zaleplon and Paracetamol), X
the concentration in ␮g ml−1 and r is the correlation coefficient.
5.5. Method validation
(a) Linearity:
5.4.1. Optimization of HPLC method
The linearity of the proposed methods was evaluated
by analyzing eight concentrations ranging between 1 and
10 ␮g ml−1 (for both D2 and 1DD spectrophotometric
methods), six concentrations ranging between 0.2 and
1 ␮g band−1 (for the spectrodensitometric method), and
nine concentrations ranging between 2 and 20 ␮g ml−1 (for
HPLC method). Each concentration was repeated three
To optimize the proposed HPLC method, all of the exper-
imental conditions were investigated. The stationary phase
choice was selected to be the reversed-phase over the normal-
phase separation due to the drawbacks of the normal phase, e.g.
hydration of silica with water which can cause peak tailing. The
optimum tailing factor was obtained when using C8 column than
Table 5
Application of standard addition technique to the analysis of Zaleplon in capsules by the spectrodensitometric method at 338 nm
Taken (␮g band−1
)
Founda (␮g band−1
)
Found %
Pure added (␮g band−1
)
Pure foundb (␮g band−1
)
Recovery %
Siesta® capsules (Batch No. 50507)
0.40
0.39
98.00
0.20
0.30
0.40
0.50
0.20
0.30
0.41
0.50
100.00
100.00
102.50
100.00
Mean S.D.
100.63 1.25
Zalocid® capsules (Batch No. 044060609/3)
0.40
0.39
97.00
0.20
0.30
0.40
0.50
0.20
0.30
0.40
0.51
100.00
100.00
100.00
102.00
Mean S.D.
100.50 1.00
a
Average of six experiments.
Average of three experiments.
b

1228
F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
Table 6
Laboratory prepared mixtures for determination of Zaleplon in presence of its
degradate by HPLC method at 232 nm
Mixture no.
Zaleplon % Taken
(␮g ml−1
Founda
(␮g ml−1
)
Found %
)
1
2
3
4
5
6
7
8
9
90
80
70
60
50
40
30
20
10
13.50
13.95
12.21
10.69
9.19
7.49
6.12
4.53
3.07
2.04
100.76
101.75
101.81
102.10
99.87
102.00
100.67
102.30
102.00
12.00
10.50
9.00
7.50
6.00
4.50
3.00
2.00
Mean S.D.
101.47 0.83
a
Average of three experiments.
times. The assay was performed according to the experi-
mental conditions previously mentioned. The linearity of the
calibration graphs and adherence of the system to Beer’s law
were validated by the high value of the correlation coefficient
and the low intercept value (Table 8).
(b) Precision:
Repeatability of the results for concentrations of 5, 7
1
and 9 ␮g ml−1 (for both D2 and DD spectrophotometric
methods), 0.3, 0.5 and 0.8 ␮g band−1 (for spectrodensito-
metric method) and 2, 7 and 12 ␮g ml−1 (for HPLC method)
were performed by three replicate determinations to estimate
intra-day variation (Table 8).
Reproducibility: Seven replicate determinations in differ-
ent 4 days were used to estimate inter-day variation. The
coefficient of variation at these concentration levels was
calculated (Table 8).
Fig. 8. HPLC chromatograms of (a) Zaleplon, (b) Paracetamol, (c) the degradate
and(d)mixtureofa, bandc. At232 nmusingacetonitrile:deionisedwater(35:65,
v/v) as a mobile phase and a flow rate of 1.5 ml min−1 using Paracetamol as
internal standard.
(c) Range:
The calibration range was established through considera-
tions of the practical range necessary according to adherence
to Beer’s law and the concentration of Zaleplon present in
the pharmaceutical products to give accurate, precise and
linear results (Table 8).
Table 7
Application of standard addition technique to the analysis of Zaleplon capsules by HPLC method at 232 nm
Taken (␮g ml−1
)
Founda (␮g ml−1
)
Found %
Pure added (␮g ml−1
)
Pure foundb (␮g ml−1
)
Recovery %
Siesta® capsules (Batch No. 50507)
5.00
4.93
98.60
3.00
5.00
10.00
13.00
3.00
5.02
9.91
100.00
100.40
99.10
12.89
99.15
Mean S.D.
99.66 0.64
Zalocid® capsules (Batch No. 044060609/3)
5.00
4.80
96.00
3.00
5.00
10.00
13.00
3.03
5.08
10.10
13.30
101.00
101.60
101
102.31
Mean S.D.
101.48 0.62
a
Average of six experiments.
Average of three experiments.
b

F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
1229
Table 8
Assay parameters and method validation obtained by applying the proposed methods for determination of Zaleplon
Parameters
Second derivative (D2)
spectrophotometric method
First derivative of ratio
spectra (1DD)
Spectrodensitometric
method
HPLC method
spectrophotometric method
Calibration range
Detection limit (LOD)
Quantitation limit (LOQ)
Slope
1–10 ␮g ml−1
0.21 ␮g ml−1
0.63 ␮g ml−1
0.0749
1–10 ␮g ml−1
0.22 ␮g ml−1
0.66 ␮g ml−1
0.2974
0.2–1 ␮g band−1
0.05 ␮g band−1a
0.2 ␮g band−1a
0.2174
2–20 ␮g ml−1
0.54 ␮g ml−1
1.64 ␮g ml−1
0.162
Intercept
Mean
0.0025
100.24
0.0248
99.9
0.0504
99.73
−0.0032
100.19
S.D.
0.86
1.07
1.35
1.15
Variance
0.74
1.14
1.83
1.32
Coefficient of variation
Correlation coefficient
R.S.D.%b
R.S.D.%c
0.009
0.9999
1.61–0.77–0.74
1.48–1.48–1.45
0.01
0.9999
0.62–0.26–0.64
0.77–1.00–0.82
0.01
0.9996
0.88–1.69–0.92
1.02–1.64–1.93
0.01
0.9998
2.00–1.25–0.82
1.63–1.63–0.82
a
Limit of detection and limit of quantitation for the spectrodensitometric method are determined experimentally.
b
R.S.D.%: the intra-day relative standard deviation of concentrations (5, 7, and 9 ␮g ml−1) for D2 and 1DD spectrophotometric (0.3, 0.5, and 0.8 ␮g band−1) for
spectrodensitometric and (2, 7, and 12 ␮g ml−1) for HPLC methods, respectively.
c
R.S.D.%: the inter-day relative standard deviation of concentrations (5, 7, and 9 ␮g ml−1) for D2 and 1DD spectrophotometric (0.3, 0.5, and 0.8 ␮g band−1) for
spectrodensitometric and (2, 7, and 12 ␮g ml−1) for HPLC methods, respectively.
Table 9
(d) Detection and quantification limits:
Statistical analysis of parameters required for system suitability testing of HPLC
method
According to the ICH recommendations [21], the
approach based on the S.D. of the response and the slope was
Parameters
Obtained value
Reference value
used for determining the detection and quantitation limits
1
(Table 8), that used for HPLC, D2 and DD spectrophoto-
Resolution (R)
3.9
R > 0.8
metric methods.
Relative retention (α)
Tailing factor (T)
2.2
1.24
2.4
>1
<1.5–2 or <2a
Capacity factor (Kꢀ)
Column efficiency (N)
1–10 acceptable
Increase with efficiency of the
separation
The smaller the value the
higher the column efficiency
S.D.
S.D.
LOD = 3.3 ×
,
LOQ = 10 ×
Slope
1648
Slope
HETPb
10−2 × 1.33
N.B.: For the spectrodensitometric method, both are deter-
mined experimentally.
a
Refers to ref. [22].
Height equivalent to theoretical plate (cm plate−1).
b
- Experimentally the detection limit is defined as the con-
centration of the analyte producing a signal which is at
least three times the base line noise measured from peak
to peak. The quantitation limit is defined as the concentra-
tion of the analyte producing the signal which is at least
ten times the base line noise.
(h) Robustness:
Different mobile phases contain water, acetonitrile,
methyl alcohol with different ratios and pH using phos-
phate buffer were tried to obtain the optimum parameters
for complete separation.
(e) Selectivity:
Selectivity of the methods was achieved by the analysis
of different laboratory prepared mixtures of Zaleplon and
its degradate within the linearity range. Satisfactory results
were shown (Tables 1, 4 and 6).
(i) System suitability testing for HPLC:
System suitability test of HPLC method gave good res-
olution (R = 3.9), relative retention time (α = 2.2), column
capacity (K = 2.4), and tailing factor (T = 1.24) (Table 9).
(f) Accuracy:
Accuracy of the methods was assured by the use of the
standard addition technique; it was performed by addition
of known amounts of pure Zaleplon to a known concen-
tration of the commercial capsules. The resulting mixtures
were assayed and the results obtained for Zaleplon were
compared with the expected results. The good recoveries of
standard addition technique (Tables 3, 5 and 7), suggested
good accuracy of the proposed methods.
6. Conclusion
It is clear from the degradation pathway of Zaleplon (Fig. 1)
that it is highly susceptible to hydrolysis. The proposed meth-
ods are efficient for providing simple, accurate and reproducible
quantitative analysis for the determination of Zaleplon in phar-
maceutical tablets, without any interference from excipients or
its degradation product.
(g) Stability:
1
Zaleplon working solutions showed no spectrophotomet-
ric, and chromatographic changes for 3 days when stored
refrigerated at 5 ◦C (stability indicating method).
D2 and DD spectrophotometric methods have the advan-
tages of low cost and speed. On the other hand, the
spectrodensitometric and HPLC methods were found to be

1230
F.H. Metwally et al. / Spectrochimica Acta Part A 68 (2007) 1220–1230
Table 10
Statistical comparison of the results obtained by the four proposed methods and the reference method
Parameters
Second derivative (D2)
First derivative of ratio
spectra (1DD)
Spectrodensitometric
method
HPLC method
Reference methoda
a
b
a
b
a
b
a
b
a
b
Mean%
S.D.
N
97.2
1.04
6
96.0
1.04
6
97.40
1.00
6
96.60
1.06
6
98.00
1.19
6
97.00
1.28
6
98.60
0.94
6
96.00
0.73
6
97.18
1.09
6
95.01
1.37
6
Variance
Student’s t-test (2.23)b
F-test (5.05)b
1.07
0.97
1.12
1.07
0.17
1.77
1.00
0.72
1.19
1.13
0.06
1.67
1.41
0.24
1.18
1.63
0.03
1.16
0.88
0.04
1.35
0.53
0.14
3.55
1.20
1.89
N.B.—a: Siesta® capsules (Batch No. 50507) and b: Zalocid® capsules (Batch No. 044060609/3).
a
Reference method is the company method.
Figure between parentheses represent the corresponding tabulated values of t and F at P = 0.05.
b
highly selective but spectrodensitometric method was found to
be the most sensitive.
excellent shelf life and available in any analytical laboratory.
These merits suggest the use of the proposed methods in routine
and quality control analysis without interference of commonly
encountered addivities.
Theproposedtechniquesaremoreselectivethanthecompany
method which is RP-HPLC using acetonitrile:water:acetic anhy-
dride (70:29.5:0.5, v/v/v) with UV detection at 280 nm using
hypersil-ODS C18 column and flow rate 1 ml min−1. Applying
the company method to the drug and its degradate, it was found
that they cannot be separated from each other, and cannot be
used for Zaleplon analysis in presence of its degradate. In addi-
tion, the proposed HPLC method has an advantage of using an
internal standard.
The results of the proposed methods were statistically com-
pared with the company method [g2] on the capsules. The t
and f values were computed (by Microsoft Excel Program) and
found to be less than the tabulated ones indicating no significant
difference with respect to accuracy and precision (Table 10).
Furthermore, statistical analysis of the results obtained by
the proposed methods and the company method were performed
with Microsoft Excel Program using one-way analysis of vari-
ance (ANOVA; F-test) at P < 0.05. The test ascertained that the
proposed methods are as precise and accurate as the company
method (Table 11).
References
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are rapid, simple and sensitive stability indicating procedures.
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Table 11
Statistical analysis of the results obtained by applying the four proposed meth-
ods and the reference method for Siesta® and Zalocid® tablets using one-way
ANOVA (F-test)
Method
Mean R.S.D.%a
[17] F. Salinas, J.J. Berzas Nevado, A. Eopinosa Mansilla, Talanta 37 (1990)
347.
a
b
[18] J.J. Berzas Nevado, J. Rodriguez Flores, M.J. Villasenor, Anal. Lett. 27
(1994) 1009.
[19] B. Morelli, Talanta 41 (1994) 673.
[20] N. Grinberg, Modern Thin-Layer Chromatography, Marcel Dekker Inc.,
New York, 1990, p. 249.
[21] The European Agency for The Evaluation of Medical Products, ICH Topic
Q2B Note for Guidance on validation of Analytical Procedures, Method-
ology GPMP/ICH/281/95, 1996.
Second derivative (D2)
First derivative of ratio spectra (1DD)
Spectrodensitometric method
HPLC method
97.20 1.07
97.40 1.03
98.00 1.21
98.60 0.95
1.9
96.00 1.08
96.60 1.10
97.00 1.32
96.00 0.76
2.62
F valueb
a: Siesta® capsules (Batch No. 50507) and b: Zalocid® capsules (Batch No.
044060609/3). n = 6.
[22] FDA’ s Center for drug Evaluation and Research (CDER), Internet:
http://pubs.acs.orgm, 5/5/2006.
a
R.S.D. = relative standard deviation.
There was no significance difference between the methods using one-way
b
ANOVA (F-test), where F tabulated = 2.76.