Bulletin of the Chemists and Technologists of Macedonia, Vol. 24, No. 2, pp.

103108 (2005)
GHTMDD 466 ISSN 0350 0136
Received: December 15, 2004 UDC: 615.214.24 : 543.544.5
Accepted: May 20, 2005
Original scientific paper
SIMULTANEOUS DETERMINATION OF DIAZEPAM AND PRESERVATIVES
IN HPMC HYDROGEL BY HPLC
1asmina Tonic-Ribarska
1
. Suzana Trajkovic-1olevska
1
. Katerina Milenkova
1
. Katerina Goracinova
2
.
Marija Glavas-Dodov
2
. Aneta Dimitrovska
1

1
Center of Drug Quality Control,
2
Institute of Pharmaceutical Technology and Biopharmacy,
Faculty of Pharmacy,Ss Cyril and Methodius University,
Vodnjanska 17, MK-1000 Skopje, Republic of Macedonia
jasminatonic@yahoo.com
Diazepam incorporated in HPMC hydrogel is administered rectally and it is used as an anticonvulsant, particu-
larly in the management of status epilepticus and febrile convulsions. The aim of this study was to develop HPLC
method for identification and simultaneous determination of diazepam and preservatives (benzyl alcohol and benzoic
acid/sodium benzoate) in HPMC hydrogel for rectal administration. Separation of these components was performed
on LiChrospher 60 RP-selected B, 125 4 mm, 5 m column, using a mixture of buffer (0.05 mol/l KH
2
PO
4
, pH 2.5)
and methanol (56 : 44 v/v) as a mobile phase. Under the experimental chromatographic conditions, satisfactory sepa-
ration of diazepam and the preservatives in hydrogel was achieved. Retention times were: 2.16, 2.66 and 8.30 min for
benzyl alcohol, benzoic acid / sodium benzoate and diazepam, respectively. Validation results showed that the
method is selective, linear, accurate and precise.
Key words: diazepam; benzyl alcohol; benzoic acid/sodium benzoate; HPMC hydrogel; simultaneous determination;
HPLC method
INTRODUCTION
Most of the pharmaceutical companies today
are oriented toward designing new pharmaceutical
dosage forms of existing drugs rather than disco-
vering new drug products. Utilization of the exist-
ing resource of marketed and patented drug sub-
stances with known therapeutic effects, and modi-
fication of their pharmaco-therapeutic characteris-
tics by incorporation in suitable drug delivery sys-
tem, has been the target of recent pharmaceutical
development [1].
Hydrogels, as a new pharmaceutical dosage
form, are now widely used. They can be used for
topical, oral and rectal drug delivery. However, in
the application of hydrogels, both the drug release
rate in vitro and the in vivo gastric transit profile
may affect in vivo usefulness [2].
Recently, hydrogel formulations have been
proposed as alternatives to conventional suppositories
and rectal solutions for the administration of various
drugs by rectal route. The use of conventional sup-
positories is accompanied by several difficulties,
such as absorption irregularity, patient discomfort
and acceptability, etc. The advantages of hydrogels
over suppositories and rectal solutions are: more
comfortable way of administration (allows easy and
safe administration at home by nonmedical person-
nel), increased stability, and prolonged duration of
action with increased therapeutic efficacy due to
viscosity of the gel matrix [3].
Diazepam incorporated in hydroxypropyl me-
thylcellulose (HPMC) hydrogel is used for rectal
application. This new rectal drug delivery system
offers an easy, safe, and bioavailable method to
administer diazepam in the management of status
epilepticus and febrile convulsions.
Diazepam is a long-acting benzodiazepine
with anticonvulsant, anxiolytic, sedative, and mus-
cle relaxant properties [4]. It is the most widely
used drug for the acute management of all types of
seizures (febrile and epileptic) in both adults and
children. Diazepam can be administered orally,
intravenously, or rectally. Rectal administration of
104 J. Tonic-Ribarska. S. Traikovic-Jolevska. K. Milenkova. K. Goracinova. M. Glavas-Dodov. A. Dimitrovska
Bull. Chem.Technol. Macedonia, 24, 2, 103108 (2005)
diazepam is the method of choice in treating and
controling acute seizures. This route of administra-
tion yields diazepam plasma levels and therapeutic
efficacy comparable to that attained when the
same dose is given intravenously [5, 6].
Since diazepam is only slightly soluble in wa-
ter, the hydrogel formulation must contain cosol-
vents for increasing diazepam solubility, such as
benzyl alcohol and benzoic acid/sodium benzoate.
These cosolvents also act as a preservatives and
buffering compounds in the formulation [7].
The official methods for determination of di-
azepam in different pharmaceutical dosage forms
are prescribed in USP 26 and BP 2002. USP 26 [8]
prescribes HPLC method for determination of di-
azepam in tablets, capsules and injections, using a
reversed-phase C18 column, a mixture of methanol
and water (65 : 35 v/v) as a mobile phase, and UV
detection at 254 nm. In BP 2002 [9], the method for
determination of diazepam in injections, tablets, oral
and rectal solutions is based on spectrophotometry.
A number of methods for the qualitative and
quantitative analysis of diazepam in pharmaceuti-
cal dosage forms have been reported, including:
UV spectrophotometry [10], derivative spectros-
copy [11], comparison of TLC and HPLC [12],
HPLC [1318], and capillary electrophoresis [19].
Various HPLC methods have been developed for
the determination of diazepam in different phar-
maceutical dosage forms. Bakavoli et al. [12] re-
ported an RP-HPLC method for determination of
diazepam in tablets using a mixture of methanol
and 0.01 M phosphate buffer, pH 7.8 (1:1 v/v) as a
mobile phase, and UV detection at 254 nm. Other
groups have also determined diazepam in tablets
using a different composition of mobile phase,
such as acetonitrile:water (30:70 v/v) [13], metha-
nol:water [14] or methanol:buffer pH 6.0 (70:30
v/v), and UV detection at 230 nm [15]. Strom et al.
[16] proposed an HPLC procedure for determina-
tion of the stability of diazepam extemporaneous
suspension compounded from tablets. Another
HPLC method was used for determination of di-
azepam in injections [17] employing mobile phase
methanol:THF:water (50:5:50 v/v) and UV detec-
tion at 254 nm.
Although many HPLC methods have been
developed for quantitative determination of diaze-
pam in various pharmaceutical dosage forms as
tablets, capsules, solutions, suspensions and injec-
tions, no HPLC method has been reported so far
for the determination of diazepam in HPMC hy-
drogel for rectal administration, or for simultane-
ous determination of diazepam and the preserva-
tives (benzyl alcohol and benzoic acid/sodium
benzoate) used in the rectal hydrogel formulation.
The aim of our study was to develop a HPLC
method for identification and simultaneous deter-
mination of diazepam and benzyl alcohol and ben-
zoic acid/sodium benzoate in this new hydrogel
drug delivery system for rectal administration.
EXPERIMENTAL
Chemicals and apparatus
All chemicals used were of HPLC or analyti-
cal grade. Diazepam was provided by FIS SpA
(Italy). The gelling agent hydroxypropyl methyl-
cellulose (HPMC-E5) was purchased from Hercu-
les (Methocel E5, USA). The antimicrobial pre-
servatives (benzyl alcohol, benzoic acid and so-
dium benzoate) and methanol were supplied from
Alkaloid (Macedonia).
The chromatographic system consisted of a Wa-
ters 600 pump, with Waters 996 photodiode array de-
tector and Millennium 32

software for data handling.

Chromatographic conditions
Samples were injected through a Rheodyne
injector valve with 20 l sample loop. Separations
were performed on Lichrospher

60 RP-select B
column, 125 4 mm, 5 m particles (Merck). A
mixture of buffer (0.05 mol/l KH
2
PO
4
, pH 2.5) and
methanol (56 : 44 v/v) was used as mobile phase.
An isocratic elution was made at a temperature of
25 C and flow rate of 1 ml/min. UV detection was
performed at 254 nm.
Preparation of diazepam rectal hydrogel
Diazepam rectal hydrogel was prepared at the
Institute of Pharmaceutical Technology at the Fac-
ulty of Pharmacy, Skopje.
Rectal hydrogel containing 2 mg/ml diazepam
was prepared by incorporation of diazepam solu-
tion in structured vehicles of HPMC. The basic
solvent in the formulation was purified water but
the solubility of diazepam in water is 0.05 mg/ml.
For that reason, the mixture of cosolvents was
used. The drug substance was dissolved in the pre-
Simultaneous determination of diazepam and preservatives in HPMC hydrogel by HPLC 105
I(ac. xc+. cxuo(. Matc0ouuja. 24. 2. 1O31O8 (2OO5)
pared mixture of cosolvents and preservatives and
it was incorporated into the structured vehicle of
HPMC by gentle mechanical stirring. The gel base
was prepared by hot/cold technique [20].
Preparation of the standard solutions for the
HPLC determination
Stock solutions were prepared by dissolving
each standard substance in methanol with concen-
trations of 0.1 mg/ml diazepam, 3.75 mg/ml benzyl
alcohol, 0.25 mg/ml benzoic acid, and 0.50 mg/ml
sodium benzoate. The mixture of these compo-
nents was prepared using the appropriate quanti-
ties of each stock solution and diluting with mobile
phase to the following working concentration: di-
azepam (0.05 mg/ml), benzyl alcohol (0.375
mg/ml), benzoic acid (0.025 mg/ml) and sodium
benzoate (0.05 mg/ml).
Typical calibration curves were constructed
with five blank hydrogel samples spiked with ap-
propriate amounts of each standard solution in the
range of 0.025 to 0.075 mg/ml for diazepam, 0.187
to 0.562 mg/ml for benzyl alcohol and 0.037 to
0.112 mg/ml for benzoic acid / sodium benzoate
(50150 % of the working concentration). The
standard samples were prepared according to the
proposed extraction procedure.
Preparation of the sample solution for the HPLC
determination
One ml (accurately measured) of diazepam
rectal hydrogel containing 2 mg/ml diazepam,
15 mg/ml benzyl alcohol, 1 mg/ml benzoic acid
and 2 mg/ml sodium benzoate, was transferred to a
20 ml volumetric flask. Ten ml of methanol were
added and the flask was placed in an ultrasonic
bath for 30 min. After cooling at the room tem-
perature, the flask was filled with methanol to the
volume mark, and the sample was centrifuged at
3000 rpm for 15 min. Five ml of the clear centrifu-
gate was transferred to a 10-ml volumetric flask,
and filled with mobile phase to the volume mark.
After filtration through a 0.45 m membrane filter,
an aliquot of the sample solution was injected into
the HPLC column.
Blank solution of HPMC hydrogel, without
diazepam and preservatives (empty hydrogel), was
prepared according to the same procedure.
RESULTS AND DISCUSSION
Determination of diazepam and preservatives
in rectal hydrogel requires the use of suitable sol-
vent for their extraction from the HPMC hydrogel.
For the preparation of the sample solution a new
method for extraction of diazepam and preserva-
tives from hydrogel, as a new drug delivery system
for rectal administration, was proposed.
Diazepam is very slightly soluble in water,
but it is soluble in alcohol. Benzyl alcohol is solu-
ble in water and miscible with alcohol, benzoic
acid is slightly soluble in water and freely soluble
in alcohol, and sodium benzoate is freely soluble
in water and soluble in alcohol [21].
Based on existing literature data, methanol
was used as an extracting solvent, to ensure com-
plete extraction of the required components from
the hydrogel. After extraction of the analytes from
the matrix and from the spiked samples, the recov-
ery values obtained from the HPLC determination
confirmed the completeness of the proposed ex-
traction procedure.
In order to achieve a satisfactory separation
of diazepam, benzyl alcohol and benzoic acid/sodium
benzoate, some of the chromatographic parame-
ters, including composition and pH of mobile
phase and detection wavelengths (230 and 254
nm), were varied. Series of mobile phases contain-
ing methanol/water with different volume fractions
and methanol/buffer solutions with different pH
values (from 2 to 5) and different volume fractions
were investigated. The most satisfactory results
were obtained when the mobile phase consisted of
a mixture of buffer (0.05 mol/l KH
2
PO
4
, pH 2.5)
and methanol in volume fraction ratio of 56 : 44,
and UV detection at 254 nm.
Under these chromatographic conditions, the
obtained retention times were approximately 2.16
min for benzyl alcohol, 2.66 min for benzoic acid /
sodium benzoate and 8.30 min for diazepam.
Using the retention times for benzyl alcohol,
benzoic acid/sodium benzoate and diazepam, the
capacity factor k, selectivity factor (for ben-
zyl alcohol/benzoic acid/sodium benzoate and di-
azepam/benzoic acid/sodium benzoate), and reso-
lution factor Rs (for benzyl alcohol/benzoic acid/
sodium benzoate and diazepam/benzoic acid/sodium
benzoate) were calculated (Table 1). The obtained
values of these three parameters (1 < k <10, >1,
106 J. Tonic-Ribarska. S. Traikovic-Jolevska. K. Milenkova. K. Goracinova. M. Glavas-Dodov. A. Dimitrovska
Bull. Chem.Technol. Macedonia, 24, 2, 103108 (2005)
Rs >2) show that the proposed chromatographic con-
ditions are suitable for separation and quantification
of the analysed components. The column efficiency
was determined by the number of plates (Table 1) and
the obtained value shows a good separation efficiency
of the applied column. The precision of the system
was confirmed by determination of repeatability of
the system (RSD % 2.0%, n = 6) (Table 1).
T a b l e 1
Characteristic parameters for system suitability
of the method
Parameters Benzyl
alcohol
Benzoic acid/sodium
benzoate
Diazepam
t
R
2.16 2.66 8.30
k 0.73 1.13 5.67
1.55 5.02
N 1821 1681 2201
Rs 2.3 11.6
Repeatability
of the system
(RSD %) 0.87 0.36 0.44
t
0
(migration time, nonretaiend species) = 1.245

Validation of the method included determina-
tion of selectivity, linearity, accuracy and precision.
The chromatograms of standard solution (a),
blank solution (b) and sample solution (c) pre-
sented in Fig. 1, show that under the experimental
chromatographic conditions diazepam, benzyl al-
cohol and benzoic acid/sodium benzoate are com-
pletely separated one from each other. In the
chromatogram of blank solution there are no inter-
fering peaks at the retention times of the investi-
gated peaks. This indicates that the method is se-
lective and can be used for identification and si-
multaneous quantification of diazepam, benzyl
alcohol and benzoic acid/sodium benzoate.
Hydrogel samples spiked with different concen-
trations of the investigated standard substances were
prepared, and the linearity of the method was deter-
mined for each component separately. The calibration
curves were constructed from the peak areas versus
concentration, in the range of 50 150 % of the
working concentration. The least-squares regression
equation of the calibration curves were:
y = 50720.6 + 84571.57 c ; r
2
= 0.9996 for di-
azepam;
y = 9239.49 + 1797.89 c ; r
2
= 0.9999 for
benzyl alcohol;
y = 2557.53 + 7565.39 c ; r
2
= 0.9999 for
benzoic acid/sodium benzoate.


a)

b)

c)
Fig. 1. Chromatograms of:
a) standard solution of diazepam (0.05 mg/ml), benzyl alcohol
(0.375 mg/ml), benzoic acid (0.025 mg/ml) and sodium
benzoate (0.05 mg/ml)
b) blank solution without diazepam, benzyl alcohol, benzoic
acid and sodium benzoate (empty hydrogel)
c) sample solution of diazepam (0.05 mg/ml), benzyl alcohol
(0.375 mg/ml), benzoic acid (0.025 mg/ml) and sodium
benzoate (0.05 mg/ml)
Quantitative determination of diazepam, ben-
zyl alcohol and benzoic acid/sodium benzoate was
performed by the external standard method. Re-
peatability of the method (method precision) was
determined by performing the analytical procedure
six times using the same sample. The indicator of
repeatability is the relative standard deviation
(RSD 2.0 %). Each result is the average of two
successive injections of the sample solution. Re-
producibility of the method (inter-day precision)
was confirmed by comparing the results obtained
from six quantitative determinations on three dif-
Simultaneous determination of diazepam and preservatives in HPMC hydrogel by HPLC 107
I(ac. xc+. cxuo(. Matc0ouuja. 24. 2. 1O31O8 (2OO5)
ferent days (RSD 2.0 %). The results are pre-
sented in Table 2.
T a b l e 2
Results of the HPLC determination of diazepam,
benzyl alcohol and benzoic acid/sodium benzoate
in hydrogel, by the external standard method
Declared
mg/ml
Determined
mg/ml
Recovery
%
SD
mg/ml
RSD
%
Intra-day (n = 6)
Diazepam 2 1.99 99.96 0.02 0.40
Benzyl alcohol 15 14.96 99.73 0.12 0.83
Benzoic acid/
sodium benzoate
3 3.02 100.76 0.05 1.55
Inter-day (n = 18)
Diazepam 2 2.01 100.49 0.02 0.89
Benzyl alcohol 15 14.98 99.84 0.06 0.41
Benzoic acid/
sodium benzoate
3 2.98 99.33 0.05 1.68

The accuracy of the method was confirmed
by the standard addition method, by adding diaze-
pam, benzyl alcohol and benzoic acid/sodium ben-
zoate standard solutions in the amount of 10, 30
and 50 % over the declared values of each ana-
lysed component in the hydrogel. The results are
presented in Table 3.
T a b l e 3
Results of the determination of diazepam, benzyl
alcohol and benzoic acid/sodium benzoate in hy-
drogel by the standard addition method
Component Declared
mg/ml
Added
mg
Calculated
mg/ml
Determined
mg/ml
Recovery
%
Diazepam 2 1.99 99.55
0.2 2.19 2.21 100.91
0.6 2.59 2.58 99.61
1 2.99 3.01 100.67
15 15.11 100.73 Benzyl
alcohol
1.5 16.61 16.37 98.55
4.5 19.61 19.38 98.83
7.5 22.61 22.69 100.35
3 2.98 99.33
0.3 3.28 3.24 98.78
Benzoic
acid/sodiu
0.9 3.88 3.86 99.48
1.5 4.48 4.41 98.44

The obtained recovery values of 99.55
100.91 % for diazepam, 98.55 100.73 % for ben-
zyl alcohol and 98.44 99.48 % for benzoic
acid/sodium benzoate in hydrogel, without and
after addition of standards, indicate that the pro-
posed method is quantitative and accurate.
CONCLUSION
Diazepam rectal hydrogel is a new pharmaceu-
tical dosage form for rectal application, composed of
the active substance diazepam, and preservatives
(benzyl alcohol and benzoic acid/sodium benzoate).
In this work, a simple and rapid reverse-phase HPLC
method was developed for identification and simulta-
neous determination of diazepam, benzyl alcohol and
benzoic acid/sodium benzoate in HPMC hydrogel
for rectal administration. A new method for extrac-
tion of the investigated substances from the hy-
drogel was developed for the preparation of the
sample solution for the HPLC determination. The
validation data show that the HPLC method is se-
lective, linear, precise, and accurate, and can read-
ily be used in the analysis of these components in
this new hydrogel rectal drug delivery system.
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P e : n x e

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cnxv1ano onpenevnane;HPLC
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xoona. co vno1peoa na cxeca on nvqep (0,05 mol/l
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