Stability indicating spectrophotometric methods for quantitative determination of bromazepam and its degradation product

Article abstract of DOI:10.1016/j.saa.2020.118433

Four simple, sensitive and selective stability indicating spectrophotometric methods are presented for quantitative determination of the benzodiazepine drug; bromazepam (BMZ) and one of its reported potential impurities and degradation product; 2-(2-amino-5-bromobenzoyl) pyridine (ABP) in methanol. Method A, is isoabsorptive point coupled with D⁰ method, where good linearity was obtained by measuring the absorbance of BMZ at 264 nm (A_iso) in the concentration range of 2–25 μg mL^?1, and the absorbance of ABP at its λ_max 396 nm in concentration range of 0.5–24 μg mL^?1. Method B, is ratio subtraction; the absorbance was measured at 233 nm for BMZ using 20 μg mL^?1 of ABP, while ABP was determined directly at its λ_max 396 nm using methanol as a solvent. Method C, was based on measuring the total peak amplitude of the first derivative of the ratio spectra (DD¹) of BMZ from 301 to 326 nm using 10 μg mL^?1 of ABP as a divisor and determination of ABP at peak amplitude of 293 nm using 5 μg mL^?1 of BMZ as a divisor. In method D, ratio difference method, good linearity was achieved for determination of BMZ and ABP by measuring the differences between the amplitudes of ratio spectra at 312 nm and 274 nm and differences between the amplitudes of ratio spectra at 274 nm and 312 nm, respectively. The stability of BMZ was investigated under different ICH recommended forced degradation conditions. The suggested methods were then successfully applied for determination of BMZ in its pharmaceutical formulations.

Full text of DOI:10.1016/j.saa.2020.118433

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 238 (2020) 118433
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Stability indicating spectrophotometric methods for quantitative
determination of bromazepam and its degradation product
b,f
Hany W. Darwisha,b, Nesma A. Ali c, Ibrahim A. Naguib d,e, , Mohamed R. El Ghobashy
,
⁎
Abdullah M. Al-Hossaini a, Maha M. Abdelrahman d
a
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia
Analytical Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini St., Cairo 11562, Egypt
Toxicology Laboratory, Forensic Medicine Authority, Justice Ministry,114 Bairam El Tounsy St., El Sayeda Zeinab, 11647 Cairo, Egypt
b
c
d
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Alshaheed Shehata Ahmad Hegazy St., 62514 Beni-Suef, Egypt
Department of Pharmaceutical Chemistry, College of Pharmacy, Taif University, Al-Hawiah 21974, Taif, Saudi Arabia
Faculty of Pharmacy, October 6 University, October 6 City, Giza, Egypt
e
f
a r t i c l e i n f o
a b s t r a c t
Article history:
Four simple, sensitive and selective stability indicating spectrophotometric methods are presented for quantita-
tive determination of the benzodiazepine drug; bromazepam (BMZ) and one of its reported potential impurities
and degradation product; 2-(2-amino-5-bromobenzoyl) pyridine (ABP) in methanol. Method A, is isoabsorptive
point coupled with D0 method, where good linearity was obtained by measuring the absorbance of BMZ at
264 nm (Aiso) in the concentration range of 2–25 μg mL−1, and the absorbance of ABP at its λmax 396 nm in con-
centration range of 0.5–24 μg mL−1. Method B, is ratio subtraction; the absorbance was measured at 233 nm for
BMZ using 20 μg mL−1 of ABP, while ABP was determined directly at its λmax 396 nm using methanol as a solvent.
Method C, was based on measuring the total peak amplitude of the first derivative of the ratio spectra (DD1) of
BMZ from 301 to 326 nm using 10 μg mL−1 of ABP as a divisor and determination of ABP at peak amplitude of
293 nm using 5 μg mL−1 of BMZ as a divisor. In method D, ratio difference method, good linearity was achieved
for determination of BMZ and ABP by measuring the differences between the amplitudes of ratio spectra at
312 nm and 274 nm and differences between the amplitudes of ratio spectra at 274 nm and 312 nm, respectively.
The stability of BMZ was investigated under different ICH recommended forced degradation conditions. The sug-
gested methods were then successfully applied for determination of BMZ in its pharmaceutical formulations.
© 2020 Published by Elsevier B.V.
Received 10 February 2020
Received in revised form 27 April 2020
Accepted 27 April 2020
Available online xxxx
Keywords:
Bromazepam
2-(2-amino-5-bromobenzoyl) pyridine
Isoabsorptive point
Ratio subtraction
First derivative of ratio spectra
Ratio difference
1. Introduction
compounds activation of gamma amino butyric acid (GABA) recep-
tors within the brain [3]. BMZ has been used for short-term treat-
The quality of a pharmaceutical product is important for patient
safety. The presence of impurities or degradation products may in-
fluence the efficacy and safety of the drug when given to a patient.
Many impurities and potential degradation products that maybe
present pharmaceutical product has the potential to cause changes
within the chemical, pharmacological or toxicological properties of
a drug, and thereby effect the product quality and safety [1]. Similar
to most compounds belonging to the 1,4- benzodiazepine series
such as diazepam, BMZ is shown to have sedative, hypnotic, muscle
relaxant, anticonvulsant and amnesic properties [2]. In general, the
inhibitory action of benzodiazepines on the CNS is a result of the
ment of anxiety disorders occurring alone or associated with
insomnia [4]. It is one of the most commonly prescribed drugs for
the treatment of anxiety and insomnia [5,6]. The assay method for
BMZ reported in the British Pharmacopeia (BP), is by anhydrous ti-
tration of BMZ by perchloric acid in the presence of acetic anhy-
dride with potentiometeric detection of end point for its assay in
pure form [7].
Several analytical methods have been reported for benzodiaze-
pines determination in pharmaceutical and biological fluids
[8–10]. There are number of publications for the analysis and deter-
mination of therapeutic and toxic blood concentration of BMZ
[11,12]. Many publications can be found that have introduced vari-
ous different analytical methods for the determination of BMZ ei-
ther individually or in combination with other benzodiazepines in
pharmaceutical and/or biological fluids including spectrophotome-
try [13–19], spectrofluorimetry [13,20], spectroscopy [21,22], HPLC
[23–39], GC–MS [40–43], TLC [44], capillary electrophoresis
⁎
Corresponding author at: Pharmaceutical Analytical Chemistry Department, Faculty of
Pharmacy, Beni-Suef University, Alshaheed Shehata Ahmad Hegazy St., 62514 Beni-Suef,
Egypt.
E-mail addresses: i.abdelaal@tu.edu.sa, Ibrahim.yaseeen@pharm.bsu.edu.eg
(I.A. Naguib).
https://doi.org/10.1016/j.saa.2020.118433
1386-1425/© 2020 Published by Elsevier B.V.

2
H.W. Darwish et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 238 (2020) 118433
[45,46], ion-selective electrode [47,48], potentiometric [49] and
voltammetric [15,50] methods.
to make the working solutions of BMZ and ABP with concentrations
equal to 100 μg mL−1
.
BMZ, similar to most benzodiazepine derivatives, can undergo
hydrolysis in acidic aqueous solutions, leading to the formation of
degradation products. Because of the BMZ extensive use, a study
on the mechanism and kinetics of its hydrolysis is a matter of
great concern. Some publications were showed interested in study-
ing the stability of BMZ, especially hydrolysis with formation of its
degradate; the fluorescent benzophenone derivative; 2-(2-amino-
5-bromobenzoyl) pyridine (ABP) [21,39,51]. The BP [7] recognizes
ABP as a potential impurity in BMZ pure substance. BMZ main met-
abolic route involves hydroxylation at C3 and hydrolysis of the het-
erocyclic ring yielding two known metabolites ABP and 3-hydroxy-
bromazepam, that reported to be eliminated by renal excretion in
urine [15,52,53].
2.4. Acidic-induced degradation and preparation of degradation product
(ABP)
As reported elsewhere [39], pure BMZ (1 g) was dissolved into the
least amount of methanol. This was followed by the addition of 1 N
HCl (50 mL). The mixed solution was then kept away from light and
refluxed for 3 h. TLC was used to monitor the reaction, with a 4:1
chloroform-acetone mixture as the development solvent. The complete
fading of the drug spot (BMZ) and the appearance of a new spot (ABP)
on the TLC plate was an indication of BMZ transformation into ABP. The
acid degradation reaction was neutralized by the dropwise addition of
2 N NaOH solution till the formation of a yellowish precipitate of degra-
dation product ABP, and then refrigerated overnight. The ABP precipi-
tate was filtered and washed several times with distilled water and
then oven-dried at 70 °C. Confirmation was obtained by the used of in-
frared and mass spectrometry.
The aim of the work, is to establish a study on the stability of
BMZ as the current ICH guidelines requirement. Stability study
should be done using developed and validated stability indicating
assay methods after stress testing on the drug under a variety of
conditions [54,55].
The proposed methods in the presented work have the advan-
tages over previously published methods of being simple, more ac-
curate and sensitive. There are few reported spectrophotometric
methods for the determination of BMZ and ABP that mainly depend
on chelation of BMZ with metals followed by measuring the absor-
bance. These methods are either time consuming or less sensitive
[14–18].
The work here presents the development and validation of a se-
lective, sensitive, and rapid, stability-indicating spectrophotomet-
ric methods, for determination of BMZ as a raw material or in
tablets, which is necessary for quality control work. In addition to
the determination of BMZ in presence of its degradation product
ABP; which is being one of its potential impurities [7] and its
main benzophenone metabolite, the spectrophotometric methods
presented here also can be used for the quantitation of ABP with
high accuracy despite the presence of high structural similarity be-
tween them.
2.5. Alkaline-induced degradation
Pure BMZ was weighted (0.50 g) and then dissolved into the
least amount of methanol, followed by the addition of 1 N NaOH
(50 mL). Then solution was protected from light and refluxed for
10 h. Similar to acidic-induced degradation procedure, the
alkaline-induced degradation reaction was also monitored by TLC
and 4:1 chloroform-acetone mixture was used as the development
solvent. When compared to acidic-induced degradation method,
both show the production of the same degradation produced
(ABP), however ABP was produced by alkaline-induced degradation
method at a slower rate.
2.6. Oxidative degradation
Compared to either an acid-induced or alkaline-induced degrada-
tion method of BMZ, the rate of ABP production by oxidative degrada-
tion of BMZ was notably slower. Oxidative degradation of BMZ was
performed by placing a weighted amount of BMZ (0.50 g) into a
10 mL conical flask containing 30% w/v solution of H2O2. The mixture
was then placed over a water bath at a controlled temperature of
80 °C for 12 h. As before, the formation of ABP by oxidative degradation
of BMZ was also followed by TLC and 4:1 chloroform-acetone solution
as the development solvent.
2. Experimental
2.1. Instrument
Spectrophotometric analysis was performed using a SHIMADZU
double beam UV–visible spectrophotometer model UV-1601 PC
(SHIMADZU, Japan), equipped with a 1 cm quartz cell. UV–PC personal
software version 3.7 was used for data collection. The spectral band
width is 1 nm and wavelength-scanning speed 2800 nm min−1. Sonix
TV ss-series ultrasonicator (USA) was used.
3. Procedure
3.1. Spectral characteristics of BMZ and ABP
The absorption spectra of BMZ and ABP (10 μg mL−1 each), and 1:1
mixture of BMZ and ABP (containing 5 μg mL−1 of each) in methanol,
was collected for isoabsorptive point, ratio subtraction methods, ratio
spectra for ratio difference method, and first derivative of ratio spectra
for DD1 method were recorded over the range 200–450 nm, by which,
methanol was used as a blank. The overlaid spectra were observed for
the selection of the most suitable wavelengths for each of the developed
methods.
2.2. Material and reagents
Bromazepam standard (purity 99.91%, according to the Certifi-
cate of Analysis) was kindly donated by Egyptian International
Pharmaceutical Industries Company (Cairo, Egypt). Lexotanil® tab-
lets (containing 3 mg BMZ per tablet) manufactured by La Roche S.
p.A. Milan, Italy, batch No. (M1139B01) and Calmepam® tablets
(containing
3
mg BMZ per tablet) manufactured by
3.2. Creation of calibration curves for isoabsorptive point coupled with D0
spectrophotometric method
GlaxoSmithKline SAE. (Cairo, Egypt), batch No. (A506716) were
purchased from a local market. HPLC grade methanol was pur-
chased from E. Merck (Darmstadt, Germany).
Accurately measured aliquots of BMZ and ABP working solutions of
equivalent to 20–250 μg of BMZ and 5–240 μg of ABP were transferred
into a divided set of 10 mL volumetric flasks, each flask was completed
with methanol. The zero order absorbance of each set was recorded
then the absorbance was measured at λ 264 nm (Aiso) for BMZ and
2.3. Standard solutions
A Stock solution of BMZ and ABP (1 mg mL−1) were prepared in ab-
solute methanol. These solutions were then further diluted in methanol

H.W. Darwish et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 238 (2020) 118433
3
λ
max 396 nm for ABP. At selected wavelength, calibration curves were
For the ratio subtraction spectrophotometric method, the absorp-
tion spectra of the laboratory prepared mixtures were divided by
the spectrum of 20 μg mL−1 of ABP (as divisor) to obtain division
spectra. The amplitude value in the plateau region at λ above
350 nm (the constant) was subtracted from the division spectra,
and then the obtained curves were multiplied by the spectrum of
20 μg mL−1 of ABP. The obtained curve used for direct determina-
tion of BMZ at 233 nm and the concentration was calculated from
its corresponding regression equation.
The procedures mentioned under creation of calibration curves
for isoabsorptive point, 1DD and ratio difference spectrophotomet-
ric methods were followed and the concentrations of BMZ and
ABP were calculated from their corresponding regression
equations.
created concerning the absorbance of each component to its corre-
sponding concentration, and then the corresponding regression equa-
tions were calculated.
3.3. Creation of calibration curves for ratio subtraction spectrophotometric
method
Accurately measured aliquots containing 10–250 μg of BMZ and
5–240 μg of ABP were transferred from their corresponding working so-
lutions (100 μg mL−1), into two separate sets of 10 mL volumetric flasks.
All of the volumetric flasks were then completed with methanol. The
zero order absorbance of each set was recorded, then the absorbance
at λmax 233 nm for BMZ and λmax 396 nm for ABP were measured. Fi-
nally, calibration curves were created by plotting the concentration of
each compound verses the absorbance of each component at the se-
lected wavelength, from which the corresponding regression equations
were obtained.
3.7. Determination of BMZ in pharmaceutical formulations (Lexotanil® and
Calmepam® tablets)
A fine powder from Lexotanil® and Calmepam® tablets were
produced, by initially weighing and then crushing 30 Lexotanil® ta-
bles and 30 Calmepam® tablets, separately. An accurately weighed
portion of each, that were equivalent to 50 mg of BMZ was trans-
ferred into two separate volumetric flasks, and then a volume of
30 mL methanol was added to each flask. The solutions were soni-
cated for approximately 30 min. Each of the prepared solitons
where then filtered into two separate 50 mL volumetric flasks. The
residual powder was washed with methanol, and finally each volu-
metric flask was completed to the mark with methanol to obtain a
stock solution with a concentration of 1 mg mL−1. The detail proce-
dures shown under the sections of creation of calibration curve for
each method was followed. Where then concentrations of BMZ
were calculated using the corresponding regression equations.
When carrying out the standard addition technique, different
known concentrations of pure standard BMZ powder were added
to certain weight of either powder of Lexotanil® or Calmepam®
tablets, and mixed well together, before proceeding in the above
mentioned procedures.
3.4. Creation of calibration curves for 1DD spectrophotometric method
Accurately measured aliquots containing 20–280 μg of both BMZ
and ABP were transferred from their corresponding working solu-
tions (100 μg mL−1) into two separate sets of 10 mL volumetric
flasks, then volume was completed with methanol. The zero order
absorption spectra of previous solutions of BMZ were divided by
the absorption spectrum of 10 μg mL−1 of ABP (as divisor) for de-
termination of BMZ. The zero order absorption spectra of ABP
were divided by the absorption spectrum of 5 μg mL−1 of BMZ (as
divisor) for determination of ABP. The obtained ratio spectra in
each case were differentiated with respect to wavelength using
Δλ = 4 nm and scaling factor = 10. The 1DD curves were recorded
and peak to peak amplitudes at 301 and 326 nm for BMZ and peak
amplitude at 293 nm for ABP were measured. Calibration curves
were created showing the relationship between the measured am-
plitudes and their corresponding concentrations of the components,
where by regression equations were obtained.
3.5. Creation of calibration curves for ratio difference spectrophotometric
method
4. Results and discussion
4.1. Elucidation of BMZ degradation product' structure (ABP)
Accurately measured aliquots containing 10–250 μg of both
BMZ and ABP were transferred from their corresponding working
solutions (100 μg mL−1) into two separate sets of 10 mL volumet-
ric flasks, then volume was completed with methanol. The zero
order absorption spectra of BMZ were divided by the absorption
spectrum of 20 μg mL−1 of ABP (as divisor). The zero order absorp-
tion spectra of ABP were divided by the absorption spectrum of
10 μg mL−1 of BMZ (as divisor). The differences between the am-
plitudes of ratio spectra at 312 nm, 274 nm and 274 nm, 312 nm
were recorded for determination of BMZ and ABP, respectively.
The calibration curves were constructed by plotting the difference
between the amplitudes of ratio spectra at the two selected wave-
lengths for each component versus the corresponding concentra-
tions and from which the corresponding regression equations
were computed.
Degradation pathway of BMZ has been identified by the litera-
ture [39]. BMZ when subjected to hydrolysis under acidic-stress
condition (in 1 N HCl solution and refluxing with for 3 h) produces
its degradation product due to breakage of the 4,5-azomethine
bond followed by cleavage of the 1,2-amide bond to give the benzo-
phenone derivative; 2-(2-amino-5-bromobenzoyl) pyridine (ABP)
and glycine, Fig. 1.
Also, when BMZ is subjected to hydrolysis under alkaline-stress
condition (refluxing with 1 N NaOH solution for 3 h) or oxidative
degradation (with 30% solution of H2O2 for 3 h) the same degrada-
tion product ABP was produced but taken more time for complete
degradation compared to acidic hydrolysis, the degradation product
was isolated as mentioned before and characterized by IR- and MS-
spectrometry.
The IR spectrum of BMZ shows two characteristic bands at about
1695.12 and 3449.06 cm−1 indicating the presence of the carbonyl
group and the –NH of the amide link, respectively. The IR spectrum
of ABP shows appearance of forked peak of –NH2 at 3420.14 and
3316.95 cm−1 which formed due to cleavage of the amide group.
Moreover, the exocyclic carbonyl group peak at 1695.12 shifted to
1623.77 due to the breakage of the ring as shown in Fig. 2(a and
b). Mass spectra were collected to give a confirmation of the IR in-
terpretations. Where the mass spectrum of BMZ (Fig. 3a) shows
parent peak identified at m/z 317 (corresponding to the molecular
3.6. Analysis of laboratory prepared mixtures of BMZ and ABP
Into 10 mL volumetric flasks, accurately aliquots equivalent to
30–200 μg of BMZ and 5–70 μg of ABP were transferred from their
working solutions (100 μg mL−1), as before each of the volume
was also completed with methanol. The absorbance of the resulting
solutions was measured at λmax = 396 nm corresponding to the
concentration of ABP alone; then ABP concentration was deter-
mined in each mixture using its corresponding regression equation.

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H.W. Darwish et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 238 (2020) 118433
Fig. 1. Degradation pathway of Bromazepam to its acidic (or alkaline) degradation product (ABP).
weight of BMZ). However, mass spectrum of ABP (Fig. 3b) formed
under acidic and alkaline hydrolysis shows a parent peak at m/z
276 (corresponding to the molecular weight of ABP).
Fig. 4, shows an overlap of the zero order absorption spectra of BMZ
and ABP, causing that direct determination of BMZ in presence of its
degradation product is extremely difficult. In the presented work, selec-
tive and sensitive determination of both BMZ and ABP could be
achieved by applying isoabsorptive point spectrophotometric method
and three methods manipulating ratio spectra (ratio subtraction, first
derivative of ratio spectra and ratio difference) where the problem of
overlapped spectra of BMZ and ABP was resolved, in addition to advan-
tages of minimal sample and data manipulation. ABP could be deter-
mined at its λmax 396 nm without any interference from BMZ.
4.2. Isoabsorptive point coupled with D0 spectrophotometric method
In this section, an isoabsorptive point spectrophotometric
method which was developed by Erram and Tipnis [56–58], was
Fig. 2. (a) IR spectrum of Bromazepam (BMZ) (b) IR spectrum of 2-amino-5-bromobenzoylpyridine (ABP).

H.W. Darwish et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 238 (2020) 118433
5
Fig. 3. (a) Mass spectrum of Bromazepam (b) Mass spectrum of 2-amino-5-bromobenzoylpyridine (ABP).
Fig. 4. Zero order absorption spectra of 10 μg mL−1 of both BMZ (—) and ABP (- - - -) and 1:1 mixture (……) containing 5 μg mL−1 of each component using methanol as a blank.
applied for simultaneous and direct determination of BMZ and its
degradation product ABP as a stability indicating assay in zero
order spectra without needing any derivatization. The theory of
isoabsorptive point spectrophotometric method could be confirmed
experimentally by recording the absorptive spectra of 10 μg mL−1 of
BMZ and ABP separately, and that of a mixture containing equal
concentration of BMZ and ABP (5 μg mL−1 of each), as shown in
Fig. 4.
By applying the suggested procedure, the absorbance at λ
264
nm (Aiso) for BMZ was obtained over different ranges, while the
concentration of ABP in BMZ and ABP mixture could be calculated
at its λmax 396 nm without any interference from BMZ. Accord-
ingly, the concentration of BMZ could be calculated by
subtraction.
Linear correlations were obtained between absorbance at
264 nm for BMZ and its concentration in the range of
Table 1
Regression and analytical parameters of the proposed methods for determination of BMZ and ABP.
Parameter
Isoabsorptive point coupled with
D0 method
Ratio subtraction method
1DD method
BMZ
Ratio difference method
BMZ
ABP
BMZ
ABP
BMZ
ABP
Calibration range (μg mL−1
Slope
Intercept
)
2–25
0.5–24
0.0220
0.0021
0.9999
100.22
100.03
1–25
2–28
2–28
1–25
1–25
0.0395
−0.0081
0.9999
100.27
100.50
0.1176
0.0186
0.9999
99.65
100.95
0.0473
−0.0013
0.9999
100.32
100.93
0.0097
−0.0001
0.9999
99.90
0.2479
−0.0239
0.9999
100.04
100.25
0.0989
0.0087
0.9999
99.63
Correlation coefficient (r)
Accuracy (mean
Specificity (mean
Precision
SD)
SD)
1.15
1.21
0.96
0.92
1.13
1.44
1.04
1.65
1.07
1.70
0.96
1.80
0.95
1.92
101.63
100.49
Repeatability (RSD%)aa
0.71
1.03
0.25
0.76
0.88
1.24
0.15
0.45
0.52
0.99
0.30
0.92
0.76
0.99
0.31
0.95
0.89
1.55
0.52
1.57
0.73
0.95
0.32
0.96
0.76
1.27
0.27
0.82
Intermediate Precision (RSD%)ba
b
LOD (μg mL−1
LOQ (μg mL−1
)
)
b
a
(RSD%)a and (RSD%)b; the repeatability and intermediate precision relative standard deviation of concentrations (5, 8, 10 μg mL−1) for BMZ and ABP.
Limit of detection and quantitation are determined via calculations (LOD = 3.3 × SD/slope, LOQ = 10 × SD/slope) [64].
b

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H.W. Darwish et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 238 (2020) 118433
Fig. 5. Absorption spectra of BMZ and ABP mixture (—), mixture after division by the divisor (-.-.-.-), mixture after subtraction of the constant (…..) and mixture after multiplication by the
divisor (- - - -) using methanol as a blank.
2–25 μg mL−1 and at 396 nm for ABP and its concentration in the
range of 0.5–24 μg mL−1, Table 1, from which the regression equa-
tions were calculated.
standard ABP (5, 10, 15 and 20 μg mL−1) were tried as divisor.
The divisor concentration of 20 μg mL−1 was found to be the best
regarding accuracy and precision when used for calculation of
BMZ concentration in the laboratory prepared mixtures. Second,
the spectra of the binary mixtures of BMZ and ABP were recorded
then divided by the chosen divisor of standard ABP. After subtrac-
tion of the amplitude value in the plateau region of the obtained
spectra above 350 nm, the obtained spectra were multiplied by
the spectrum of the divisor (20 μg mL−1) as shown in Fig. 5. Finally,
the last spectra were used for direct determination of BMZ at
233 nm.
4.3. Ratio subtraction spectrophotometric method
Ratio subtraction spectrophotometric method [59,60] proposed
for determination of BMZ; is applied in the following sequence.
First, the zero order spectra of standard solutions of BMZ were re-
corded, then the linearity of BMZ was established in concentration
range 1–25 μg mL−1 at λmax 233 nm. Different concentrations of
Fig. 6. First derivative of ratio spectra of BMZ (2–28 μg mL−1) (—) and 5 μg mL−1 of ABP (- - - -) using 10 μg mL−1 of ABP as a divisor and methanol as a blank.
Fig. 7. First derivative of ratio spectra of ABP (2–28 μg mL−1) (—) and 20 μg mL−1 of BMZ (- - - -) using 5 μg mL−1 of BMZ as a divisor and methanol as a blank.

H.W. Darwish et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 238 (2020) 118433
7
Fig. 8. Ratio spectra of BMZ (1–25 μg mL−1) (—) and 10 μg mL−1 of ABP (- - - -) using 20 μg mL−1 of ABP as a divisor and methanol as a blank.
Calibration curve was created relating absorbance at 233 nm
overlapped spectra without prior separation; meanwhile it doesn't re-
for BMZ and its concentration in the range of 1–25 μg mL−1
,
quire any sophisticated apparatus or computer programs [62,63].
The absorption spectra of BMZ in the range of 1–25 μg mL−1 were di-
vided by the absorption spectrum of a chosen concentration of
20 μg mL−1 of ABP as a divisor and the absorption spectra of ABP in
the range of 1–25 μg mL−1 were divided by the absorption spectrum
of 10 μg mL−1 of BMZ as a divisor to get the new ratio spectra as
shown in Figs. 8 & 9. Good linearity was achieved for determination of
BMZ and ABP by plotting the differences between the amplitudes of
ratio spectra at 312 nm, 274 nm and differences between the ampli-
tudes of ratio spectra 274 nm, 312 nm, respectively, versus the corre-
sponding concentrations.
Results of assay validation are demonstrated in Table 1; where the
data reveal that the methods are accurate, precise and specific over
the specified ranges. Recovery studies were performed by analyzing lab-
oratory prepared mixtures of BMZ and ABP prepared in different ratios
to demonstrate the validity and applicability of the proposed spectro-
photometric methods. Results obtained are shown in Table 2. The pro-
posed methods were successfully applied for determination of BMZ in
the pharmaceutical formulations Lexotanil® and Calmepam® tablets
and by applying the standard addition technique, no interference due
to excipients and additives was observed as shown in Table 3 for analy-
sis of BMZ in its pharmaceutical formulations.
from which the regression equation was obtained.
4.4. 1DD spectrophotometric method
The first derivative of the ratio spectra method was applied to solve
the difficulty caused by the overlapping spectra of BMZ and ABP, with
main advantage of cancelling the whole spectrum of interfering sub-
stance [61]. Selection of divisor concentration is of great importance,
so several divisor concentrations (5, 10, 15 and 20 μg mL−1) of both
BMZ and ABP were tried. The best results in terms of signal to noise
ratio, sensitivity and selectivity were obtained using 10 μg mL−1 of
ABP and 5 μg mL−1 of BMZ as divisors for determination of BMZ and
ABP, respectively.
The absorption spectra of BMZ in the range of 2–28 μg mL−1 were di-
vided by the absorption spectrum of 10 μg mL−1 of ABP and the absorp-
tion spectra of ABP in the range of 2–28 μg mL−1 were divided by the
absorption spectrum of 5 μg mL−1 of BMZ. The obtained ratio spectra
were differentiated with respect to wavelength using Δλ = 4 nm and
scaling factor = 10 where the interference in each case was eliminated
as shown in Figs. 6 and 7. Peak amplitude values of 1DD spectra showed
good linearity for determination of BMZ and ABP by measuring peak to
peak amplitudes at 301 and 326 nm and peak amplitudes at 293 nm, re-
spectively in their concentration ranges.
5. Method validation
For the proposed methods; method validation was accomplished ac-
cording to the guidelines of the international conference on harmoniza-
tion (ICH) [64]. The results of accuracy, repeatability and intermediate
precision of the methods, are found in Table 1. Linearity of the calibra-
tion graphs and adherence of the system to Beer's law were validated
4.5. Ratio difference spectrophotometric method
The most important feature of the ratio difference method is its sim-
plicity, rapidity and accuracy besides ability to solve severely
Fig. 9. Ratio spectra of ABP (1–25 μg mL−1) (—) and 20 μg mL−1 of BMZ (- - - -) using 10 μg mL−1 of BMZ as a divisor and methanol as a blank.

8
H.W. Darwish et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 238 (2020) 118433
by the high value of the correlation coefficient and the intercept value.
The good recoveries of pure drug samples suggest good accuracy of
the proposed methods, Table 1.
For the developed spectrophotometric methods presented here, we
can find that the results obtained here were statistically compared to
those obtained by applying the reported HPLC method [25] for
Lexotanil® and Calmepam® tablets. No significant difference regarding
accuracy and precision between the results was obtained. Table 4 shows
that the calculated t- and F- values were less than the theoretical values.
Moreover, one-way ANOVA test, was applied for statistical analysis of
the results obtained, by all of the proposed methods and the reported
one, showing no significant difference between them as given in
Table 4.
6. Conclusion
In conclusion, the described methods give accurate and precise re-
sults for determination of BMZ and ABP. The presented spectrophoto-
metric methods can be regarded as
a useful alternative to
chromatographic techniques in the routine quality control (QC) analysis
of BMZ pharmaceutical formulations, allowing quantitative measure-
ments to be simple and rapid at relatively low costs. These methods
are suggested to be used, due to its simplicity and low cost, in some
poor and low developed countries for QC screening of pharmaceutical
formulations containing BMZ, instead of more expensive methods
such as HPLC, which may not always be available due to the instruments
high cost, and requirement of large volumes of solvents for HPLC
analysis.
The proposed isoabsorptive point coupled with D0 method has
advantages of being sensitive and selective for determination of
BMZ due to its handling of zero order spectrum of the drug, addi-
tionally, minimal sample and data manipulation compared to
other published spectrophotometric methods which are time con-
suming or less sensitive. Three spectrophotometric methods ma-
nipulating ratio spectra (ratio subtraction, first derivative of ratio
spectra and ratio difference) provided selective and sensitive de-
termination where the problem of overlapped spectra of BMZ and
ABP was completely resolved without any prior separation. More-
over, they have advantage of being the first stability indicating
spectrophotometric methods for determination of both BMZ and
ABP.
CRediT authorship contribution statement
Hany W. Darwish:Writing - review & editing.Nesma Aboelyazeed:
Data curation, Writing - original draft.Ibrahim A. Naguib:Conceptuali-
zation, Methodology, Supervision.Mohamed R. El Ghobashy:Conceptu-
alization, Methodology, Supervision.Abdullah M. Al-Hossaini:Writing -
review & editing.Maha M. Abdelrahman:Conceptualization, Methodol-
ogy, Supervision.
Declaration of competing interest
Authors would like to declare absence of any conflicts of interest re-
garding this work. The authors declare that they have no known com-
peting financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgment
“The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at the King Saud University for funding
this work through the Research Group Project No. RGP-322”. The au-
thors would like to acknowledge Egyptian International Pharmaceutical
Industries Company (Cairo, Egypt) for their assistance in having the
pure sample of bromazepam.

H.W. Darwish et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 238 (2020) 118433
9
Table 3
Determination of BMZ and ABP in laboratory prepared mixtures by suggested spectrophotometric methods.
Degradate%
Concentrations
Recovery %a
(μg mL−1
)
Isoabsorptive point coupled with
D0 method
Ratio subtraction method
BMZ
1DD method
BMZ
Ratio difference method
BMZ
ABP
BMZ
ABP
ABP
BMZ
ABP
2.4%
5%
20
19
18
17
8
7
3
5
4
0.5
1
2
3
2
3
2
5
6
99.81
99.54
99.09
99.09
102.05
99.85
99.77
99.85
99.77
100.82
100.68
99.29
100.03
100.48
100.11
103.15
101.71
101.87
98.81
99.89
99.39
102.76
101.30
100.95
–
–
–
–
–
–
101.99
102.08
100.47
101.80
98.08
98.41
97.69
99.93
101.77
100.25
98.38
98.23
97.51
102.28
101.21
101.26
102.39
102.17
101.01
100.49
10%
15%
20%
30%
40%
50%
60%
70%
102.74
99.74
101.17
100.10
101.50
98.73
101.57
100.06
100.50
102.69
99.90
101.56
99.15
100.99
101.61
98.47
103.10
102.50
101.67
98.93
104.05
102.50
101.00
99.64
102.76
101.63
3
7
Mean
S.D
1.21
0.92
1.44
100.93
1.65
1.70
1.80
1.92
a
Average of three determinations.
Table 4
Statistical analysis of the proposed spectrophotometric methods and the reported HPLC method for determination of BMZ in Lexotanil® and Calmepam® tablets.
Pharmaceutical
formulations
Parameters
Isoabsorptive point coupled with D0
method
Ratio subtraction
method
1DD
method
Ratio difference
method
Reported HPLC
methodb
Lexotanil® tablets
(B.N. M1139B01)
Mean
SD
Variance
n
99.62
0.71
0.50
6
0.78
2.46
1.20
99.41
0.95
0.91
6
99.34
0.53
0.28
6
1.38
4.39
98.94
0.80
0.63
6
1.96
1.95
99.00
0.96
0.92
6
1.73
1.34
100.04
1.11
1.23
6
−
−
Student's t-testa (2.228)
F- testa (5.050)
Anova Fa calc. (2.53)
Mean
Calmepam® tablets
(B.N. A506716)
98.89
0.82
0.67
6
1.95
2.07
99.18
0.88
0.77
6
1.44
1.81
98.88
1.13
1.28
6
1.74
1.09
100.04
1.18
1.39
6
−
−
SD
Variance
n
Student's t-testa (2.228)
F- testa (5.050)
Anova Fa calc. (2.53)
1.02
1.53
1.30
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