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509

Simultaneous Determination of Benzodiazepines by Ultraviolet -

Visible Spectrophotornetry in Micellar Media
M. de la Guardia, M. V. Galdu, J. Monzo and A. Salvador
Departamenio de Quimica Analiiica, University of Valencia, C/Dr. Moliner 50, Burjassot 46700, Valencia,
Spain
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A method for the simultaneous determination of benzodiazepines in binary mixtures is proposed, based on
the acid hydrolysis of benzodiazepines to benzophenones and the spectrophotometric determination of the
latter in the presence of Nemol K 1030, a non-ionic surfactant condensate of ethylene oxide with nonylphenol.
The experimental conditions for the hydrolysis of several benzodiazepines in sealed Pyrex tubes were
determined. The addition of Nemol K 1030 to acidic solutions of benzophenones modified the positions of the
absorption bands and made possible the simultaneous analysis of binary mixtures of benzodiazepines.
Keywords : Benzodiazepines; benzophenones; hydrolysis; surfactants; UV spectrophotometry

Benzodiazepines are psychotherapeutic drugs widely used in Experimental

the treatment of different nervous diseases such as anxiety,
insomnia and epileptic convulsions, owing to the wide variety
of the properties of the benzodiazepinic structure. 1-3 The
therapeutic interest in these compounds justifies research to
establish analytical methods for the determination of benzo-
diazepines in pharmaceutical preparations and biological
samples.
Methods proposed for the determination of benzodiaze-
pines include liquid chromatography,"5 polarography,h,7
atomic absorption spectrometry8 and molecular spectroscopic
techniques such as f-luorimetryy.1(' and infrared" and U V -
visible spectrophotometry.
For the spectrophotometric determination of this type of
compound either the benzodiazepine itself or the benzophe-
none obtained by hydrolysis can be employed.12-13 Other
methods involve the use of colour reagents that react with
these compounds to form species that absorb in the visible
region. 14.15
The acid hydrolysis of benzodiazepines to benzophenones
has been reportedl.16 and used for the chromatographic
determination of these compounds in biological matrices. The
hydrolysis is carried out with hydrochloric acid at elevated
temperature. One of the methods proposed for obtaining
5-chloro-2-(methylamino)benzophenone(CMAB)l7 involves
the treatment of diazepam in stoppered tubes with 6~
hydrochloric for 1 h at 100°C in a boiling water-bath, but in
the paper cited the experimental conditions of the hydrolysis
reaction were not discussed. In this work we have extended
the acid hydrolysis in closed tubes to a wide variety of
benzophenones and studied the effect of various experimental
conditions.
Ultraviolet spectrophotometry is not a selective method
because many benzodiazepines have very similar spectra. The
use of organised media, such as micellar media, can improve
the sensitivity and selectivity of spectrophotometric methods
by providing hyperchromic and bathochromic shifts in the
spectra of metallic complexes and organic molecules. 18-20
Previous work has demonstrated that the addition of
anionic surfactants to solutions of some benzodiazepines in
dilute sulphuric acid improves their spectrofluorimetric deter-
mination. However, the increase in the fluorescence of
benzodiazepines in micellar media is due to the increase in the
fluorescence quantum yield without a simultaneous increase
in the molecular absorption bands.21-22
In this paper, a method is proposed for the determination of
benzodiazepines in binary mixtures based on their hydrolysis
to benzophenones and the spectrophotometric determination
of the latter in the presence of a non-ionic surfactant.
Apparatus and Reagents
A Shimadzu UVi240 U V - visible spectrophotometer
equipped with 1-cm quartz cells and a PR-1 graphic printer
was used for the absorbance measurements.
Powdered samples of diazepam, oxazepam, potassium
chlorazepate, temazepam, prazepam, nitrazepam, clonaze-
pam and lorazepam were obtained from the Clinical Hospital,
Madaus Cerafarm and the Bromatology Department of
Valencia University.
The following surfactants were used: sodium dodecyi
sulphate (SDS) (Fluka), cetyltrimethylammonium bromide
(CTAB) (Merck), Descoxid 728, a fatty acid condensate with
ethylene oxide (Tensia Surfac), Nemol K 539, 1030 and 1032
condensates of ethylene oxide with nonylphenol and Genapol
PF 80 condensate of ethylene oxide and propylene oxide
(Hoestch Iberica) and Triton X-100 condensate of tert-octyl-
phenol with ethylene oxide (Probus).
Hydrolysis of Benzodiazepines
Solutions containing 125 pg of diazepam or oxazepam in dilute
hydrochloric acid (1 + 1) were used to study the conditions for
the hydrolysis of benzodiazepines to benzophenones.
Experimental parameters such as temperature, time and acid
volume were modified using sealed Pyrex tubes to carry out
this reaction in all instances.
The recommended procedure consists of placing 1 4 ml of
the solution of a benzodiazepine or mixtures of benzodiaze-
pines in 6 M hydrochloric acid in a 15-ml Pyrex tube. The tube
is hermetically sealed and allowed to stand in an oven at
100-120 "C for 1-1.5 h , allowed to cool and then the contents
are diluted to an appropriate volume.
Diazepam and oxazepam have strong absorption bands at
240 and 235 nm, respectively, and two other common bands at
285 and 360 nm. The related benzophenones CMAB and
2-amino-5-chlorobenzophenone (ACB) both have a maxi-
mum absorbance at 260 nm (see Fig. 2 ) . The hydrolysis
reaction was monitored by spectrophotometric measurements
in the U V region.
Determination in the Presence of Surfactants
A series of cationic, anionic and non-ionic surfactants were
added to the CMAB solutions obtained after hydrolysis of
different amounts of diazepam. Calibration graphs in the
ultraviolet range were obtained in both the presence and
absence of surfactants to determine the effect on the intensity
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AKALYST, APRIL 1989, VOL. 114

0.4 -
a
c
A
4 0.3 -
C n
$
A
Q:
0.2 -

0.1 ' I I

20 60 100 140 50 80 110 140

TemperaturePC
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Fig. 2. Effect of temperature on the acid hydrolysis of diazepam and

oxazepam. ( a ) (U) Absorbance at 240 nm and (@) absorbance at 260
nm of a 5 p.p.m. solution of diazepam after treatment for 1 h with 1 ml
250 3CO 350 400 250 300 350 400 of 6~ hydrochloric acid, ( b ) (m) Absorbance at 235 nm and (@)
hln m absorbance at 260 nm of a 5 p.p.m. solution of oxazepam treated for
1 h with 1 ml of 6~ hydrochloric acid at different temperatures
Ultraviolet - visible spectra of diazepam, oxazepam and their
related benzophenones. Spectra were obtained for 5 p.p.m. solutions
of (A) diaze am and (C) oxazepam and the correspondin benzo-
phenones, 6) 5-chloro-2-(methylamino)benzophenone (SCMAB)
and (D) 2-amino-5-chlorobenzophenone (ACB)
0.4

a
and shift of the absorption bands. A 1% surfactant concentra- 0.3
tion was used in all instances except for solutions of CTAB, for 2
which a 0.05% solution was used owing to its low solubility.
2 0.2
Simultaneous Analysis of Mixtures of Benzodiazepines
I , I
Binary mixtures of benzodiazepines were hydrdysed using the 0.1 ' '
15 45 75 105 15 45
I I

experimental conditions determined previously and the UV
spectra recorded in the presence of 1% Nemol K 1030. The
absorbance values at the maximum absorbance wavelengths
of each benzodiazepine make it possible to determine the
concentration of each compound in the mixture using the
experimental data obtained for solutions containing only one
of the compounds concerned and solving simultaneous equa-
tions.
75 105
Timeimi n
Fig. 3. Effect of time o n the hydrolysis of diazepam and oxazepam.
(a) (M) Absorbance at 240 nm and (@) absorbance at 260 nm of a 5
p.p.m. solution of diazepam after treatment at 100 "C with 1 ml of 6 M
hydrochloric acid. ( b ) (U) Absorbance at 235 nm and (0)absorbance
at 260 nm of a 5 p.p.m. solution of oxazepam after treatment at 100°C
with 1 ml of 6~ hydrochloric acid

Results and Discussion

Hydrolysis of Benzodiazepines
When samples of diazepam or oxazepam are heated in an oven 0.1 I I I I

for 1 h with 1 ml of 6 M hydrochloric acid in pressurised Pyrex 0 0.5 1 0 1 2

vessels, the absorbance of the bands corresponding to
benzophenones (260 nm) increases and the absorbance of
those of the benzodiazepines (240 and 235 nm) decreases as
the temperature is increased. At temptratures higher than
100 "C a constant absorbance was obtained in both instances
(see Fig. 2). Using this temperature, the absorbance of the
benzophenones obtained by hydrolysis of diazepam and
oxazepam increases as the digestion time increases. For a time
shorter than 1 h the hydrolysis is not complete, but for longer
times the absorbance values remain constant (Fig. 3). A
volume of 6~ hydrochloric acid greater than 0.75 mi is
required for the hydrolysis of diazepam and oxazepam under
the previously determined conditions, as can be seen in Fig. 4.
Spectroscopic Characteristics of Benzophenones
Using the experimental conditions determined previously, the
hydrolysis of a series of benzodiazepines was carried out and
the following benzophenones were obtained: CMAB from
diazepam and temazepam, ACB from oxazepam and potas-
sium chlorazepate, 2-amino-S,2'-dichlorobenzophenone
(ADCB) from lorazepam, S-chloro-2-(cyclopropylmethyl-
amino)benzophenone (CCMB) from prazepam, 2-amino-5-
nitrobenzophenone (ANB) from nitrazepam and 2-amino-2'-
chloro-5-nitrobenzophenone (ACNB) from clonazepam. The
corresponding absorbance maxima and molar absorptivities
are given in Table 1. These data indicate the existence of
strong overlapping of the absorption bands corresponding to
VHcliml
Fig. 4. Effect of the volume of hydrochloric acid used on the
hydro1 sis of diazepam and oxazepam. ( a ) (H) hbsorbancc at 240 nm
and (d) absorbance at 260 nm of a 5 p,p.m. solution of diazepam
treated at 100 "C for 1 h with different amounts of hydrochloric acid.
( b ) (M) Absorbance at 235 nm and (@) absorbance at 260 nm of a 5
p.p.m. solution of oxazepam treated at 100 "C for 1 h with different
volumes of 6 M hydrochloric acid
the different benzodiazepines, which makes their differentia-
tion difficult.
Determination of Beozophenone in Micellar Media
Calibration graphs for C,MAB in both the absence and
presence of different surfactants were obtained. Table 2 gives
the equations corresponding to the systems studied. It can be
seen that the absorbance maximum at 260 nm is little affected
by the addition of anionic, cationic or non-ionic surfactants.
However, when a caticnic surfactant such as CTAB or a
non-ionic ethylene oxide condensate was added to CMAB
solution, a new absorbance band appeared at 415 nm, which
makes possible a more selective determination of this benzo-
phenone.
The addition of Nemol K 1030 to the different benzophe-
nones provides a bathochromic shift of the UV bands except
for ANB and ANCB, in all instances good linearity of the
Calibration lines corresponding to each of the benzopheno-
nones being obtained (Table 3).
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ANALYST, APRIL 1989, VOL. 114 511

The new bands obtained in acidic micellar media are the

Table 1. Spectroscopic characteristics of benzophenones same as those obtained in absolute ethanol, which indicates
that the micellar microenvironment has similar characteristics
Benzophenone hrn.3, h m &/moll-1cm I
to alcohols. In the micellar media the absorbance bands of
CMAB . . . . . . 260 11 100 k 300 some of the benzophenones derived from different benzo-
ACB . . . . . . . . 258 10300 k 100 diazepines differ significantly, in contrast to the benzodiaze-
ADCB . . . . . . . . 2.50 10 100 k 100
CCMB . . . . . . . . 260 10200 k 100
pine and benzophenone spectra obtained in acidic media,
ANB . . . . . . . . 240 13 800 f 100 hence allowing the selective determination of these com-
365 14800 k 100 pounds in binary mixtures and the determination of diazepam
ACNB . . . . . . . . 250 11 600 t 100 and its metabolites.
355 10200 k 100
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Analysis of Mixtures of Benzodiazepines

Table 2. Effect of addition of surfactants on the spectrophotometric
determination of diazepam as CMAB It can be see in Fig. 1 that diazepam and oxazepani have very
Concen- Regression
tration, Calibration coefficient
Surfactan t Yo graph* (r)
None . . . . . . - = 11.057C + 0.02 0.99995
SDS . . . . . . 1 A,,, = 10.565C + 0.01 0.99995
CTAB . . . . . . 0.05 A,,, = 11.S48C - 0.05 0.999995
A 4 1 5 = 1.819C - 0.001 0.99995 4
Nemol K 1030 .. 1 A d I 5= 3.931C + 0.006 0.999995 .?
Genapol PF80 .. 1 A260= 10.811C 0.01 + 0.999995
* A = absorbance; C = concentration in mmol 1 - 1 .
J

Table 3. Influence of Nemol K 1030 micelles on the

spectrophotometric determination of benzophenoncs 3 400 450 500

Benzo- Calibration graph Calibration graph Ah/

phenone in acidic media" in micellar media* nm
CMAB . . A,,,) = 11.057C + 0.008 A420= 4.361C + 0.004 + 160
(Y = 0.99995) ( r = 0.9995)
ACB +
. . A 2 5 8 = 10.161C 0.020 A190= 2.540C + 0.009 +I32
( r = 0.9995) ( r = 0.995)
ADCB . . AZsn= 10.13SC+ 0.020 A 3 9 5 = 4.179C - 0.003 + 145
( r = 0.99995) ( r = 0.9995)
CCMB . . A 2 6 0 = 10.093C + 0.006 A410 = 4.658C - 0.003 +I50 5 0
(Y = 0.99995) ( r = 0.99995) A/nm
ANB +
. . A T 6 5 = 14.774C-0.004 A,,,) = 14.S32C 0.01 1 -5 Fig. 5 . Absorption spectra of CMAB and ACB in acidic and micellar
(Y = 0.99995) ( r = 0.999995) media. ( a ) Absorption spectra of (1) 5 p.p.m. of CMAB; (2) 5 p.p.m.
ACNB . . A 3 5 5 = 10.238C-t 0.003 = 12.17SC + 0.011 0 of ACB; and (3) a mixture of 5 p.p.m. of both CMAB and ACB. ( b )
( r = 0.99995) (Y = 0.999995)
Absorption spectra in the presence of 1% Nemol K1030 for (4) 10
p.p.m. of CMAB; ( 5 ) 10 p.p.m. of ACB; and (6) a mixture of 5 p.p.m.
* A = absorbance; C = concentration in mmol 1 - 1 . of both CMAB and ACB

Table 4. Analysis of binary mixtures of benzodiazepines. Concentrations in p.p.m.

Relative Relative
difference, difference,
Compound Present Found Yo Compound Present Found O/*

Oxazepam . . . . . . . . 2.62 2.72 -3.8 Diazepam . . . . . . . 2.70 2.92 -8.1

5.24 5.66 -8.02 1.35 1.35 0
2.62 2.88 -9.9 2.62 2.62 0
1.31 1.10 + 16.03 5.24 5.29 -0.95
5.24 5.97 - 13.9 1.31 1.41 -7.6
1.37 1.36 +0.7 2.82 2.96 -5.0
2.74 2.99 -9.1 2.82 2.97 -5.3
5.48 6.02 -9.8 1.41 1.44 -2.1
Diazepam . . . . . . . . 1.23 1.32 -7.3 Chlorazepate .. . ., 2.54 2.70 -6.3
2.46 2.62 -6.5 2.54 2.54 0
1.23 1.31 -6.5 5.08 5.15 -1.4
4.92 5.20 -5.7 1.27 1.14 + 10.2
1.23 1.30 -5.7 2.46 2.60 -5.7
2.46 2.51 -2.03 2.46 2.66 -8.1
1.23 1.10 + 10.6 4.92 5.41 - 10.0
4.92 5.10 -3.7 1.23 1.32 -7.3
Diazepam . . . . 1.23 1.28 -4.1 Lorazepam . . .. 2.60 2.91 -11.9
2.46 2.71 - 10.2 2.60 2.44 +6.2
1.23 1.18 +4.1 5.20 5.30 -1.9
4.92 5.22 -6.1 1.30 1.30 0
1.25 1.34 -7.2 2.60 2.59 +0.4
2.50 2.73 -9.2 2.60 2.39 +8.1
1.25 1.40 - 12.0 5.20 5.02 +3.5
5.00 5.33 -6.6 1.30 1.20 +7.7

512
similar absorption spectra in the UV - visible region. The
spectra of CMAB and ACB are also very similar and as a
consequence it is not easy to determine the individual
compounds in a mixture of the two [Fig. 5(a)]. However, when
Nemol K 1030 is added to the benzophenone solutions the
absorption spectra of the two benzophenones differ substan-
tially and the binary mixture could be resolved by solving a
conventional system of simultaneous equations [Fig. 5(b)].
The same situation occurs for mixtures of diazepam and
lorazepam and diazepam and potassium chlorazepate (see
Table 3). Hence the simultaneous determination of these
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benzodiazepines in binary mixtures was carried out by
measuring the absorbance at the maximum absorption
wavelength for each compound in micellar media after
hydrolysis to the corresponding benzophenone. Table 4
summarises the results obtained and the relative differences
between the concentrations found and added.
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ANALYST, APRIL 1989, VOL. 114
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Paper 8/02781A
Received July 11th, 1988
Accepted November 2nd, 1988