A New Method for the Assay of Bisoprolol

Using Bromocresol Green

ALINA DIANA PANAINTE, NELA BIBIRE*, GLADIOLA TANTARU, MIHAI APOSTU, MADALINA VIERIU, VASILE DORNEANU
“Grigore T. Popa” University of Medicine and Pharmacy, Faculty of Pharmacy, Analytical Chemistry Department, 16 University Str.,
700115, Iasi, Romania

A new Vis spectrophotometric method was developed for the assay of bisoprolol in pharmaceutical
preparations using bromocresol green in hydrochloric acidic medium. The reaction product showed a
maximum absorbance at 402 nm proportional with the concentration of bisoprolol. The optimum conditions
for the reaction were established. The developed method was validated. The method showed a good
linearity in the concentration range of 7 – 80μg/mL (correlation coefficient r = 0.9998). The detection limit
(LOD) was 1.78 μg/mL and the quantification limit (LOQ) was 5.41 μg/mL. The precision and the accuracy
were determined; mean recovery was 100.11% in the 98.35-101.57% concentration range.
Keywords: bisoprolol, bromocresol green, spectrometric method, validation.

Bisoprolol fumarate (±)-1-{p-[(2-isopropoxyethoxy) in chloroform and then spectrophotometricaly analyzed at

methyl] phenoxy}-3-isopropyl-amino-2-propanol hemi-
fumarate is a beta1-selective (cardioselective)
adrenoceptor blocking agent without significant
membrane stabilizing or intrinsic sympathomimetic
activities in its therapeutic dose range. It is prescribed for
the treatment of hypertension and angina pectoris [1, 2].
The S(-) enantiomer is responsible for most of the beta-
blocking activity. The structural formula is presented in
figure 1.
Fig. 1. Chemical structure of
bisoprolol fumarate (2:1)
In comparison with other beta-selective blockers
(atenolol, metoprolol, betaxolol) bisoprolol proved to be
the compound with the highest beta-selectivity in all in
vitro and in vivo experiments and in all animal species
investigated [3-8].
Several methods have been reported for the
determination of bisoprolol in plasma and urine samples:
high-performance liquid chromatography (HPLC) [9–14],
liquid chromatography–tandem mass spectrometry (LC–
MS-MS) [15–18], liquid chromatography–electrospray
ionization mass spectrometry (LC–ESI-MS) [19–20] and
electrophoresis [21]. Spectrophotometric methods UV-Vis
absorption are fast, robust, precise and universally accepted
in pharmaceutical analysis [22]. Most spectrophotometric
methods for bisoprolol determination are in UV range. In
literature there are few methods for the analysis of
bisoprolol in visible domain that include reactions with
either 7,7,8,8-tetracyano qumodimethane [23], tropaeolin
000 [24] or chromazurol S [24] with maximum absorption
at 587.5 nm, 485 nm and 505 nm respectively.
The aim of the present investigation was to develop a
new rapid and reproducible method for the quantitative
determination of bisoprolol fumarate in pharmaceutical
products. The method was based on the formation of blue
coloured ion pair complex between bisoprolol and
bromocresol green in acidic medium, which was extracted
* email: nelabibire@yahoo.com
916 http://www.revistadechimie.ro
402 nm (λmax).
Experimental part
Materials and method
Absorbance was measured in quartz cuvettes using a
Hewlett Packard 8453 UV–Vis spectrophotometer at the
room temperature. An ultrasonic bath and a vibration shaker
IKA-Werke type VX2 were also used.
The reagent used were: bisoprolol fumarate – 100.07%
pure reference substance, Unichem Laboratories LTD,
India; chloroform - Fluka, Germania; hydrochloric acid -
Tunic Prod, Romania; bromocresol green - Tunic Prod,
Romania.
The pharmaceutical products Concor® (Merck),
Bisotens® (Antibiotice Iasi) and Bisoblock® (Keri Pharma
Generics Ltd) have been purchased from the local
pharmacies.
A 100 μg/mL stock bisoprolol solution was prepared
and diluted to obtain standard solutions of various
concentrations.
In order to establish the optimum working conditions,
two solutions of 7 μg/mL and 80 μg/mL were used (the
minimum and maximum concentration of the linear
range), while the parameters of the method were modified.
The optimum concentration of bromocresol green solution
and hydrochloric acid solution were established. The most
suitable solvent has been established for the extraction.
The stability of the reaction product was studied. The
validation of the proposed method was done according to
ICH guidelines [25]. The following parameters have been
studied: linearity, detection and quantification limit, system
method precision, the accuracy and robustness of the
method [26-29].
Procedure for pharmaceutical formulation
The developed method was applied for the assay of
bisoprolol in three pharmaceutical products. Twenty
bisoprolol tablets for each product were crushed into a
mortar, then a quantity of powder equivalent to 5 mg
bisoprolol was stirred in 50 mL methanol on an ultrasonic
bath. Afterwards the solution was filtered and then it was
evaporated to dryness. The residue was dissolved into a
REV. CHIM. (Bucharest) ♦ 65♦ No.8 ♦ 2014

100 mL volumetric flask using distilled water. A 20 μg/mL
solution was obtained through dilutions. Various
concentrations were obtained through dilution with distilled
water. Then 1 mL sample was processed according to the
working procedure.
Assay procedure
1 mL 0.1 M hydrochloric acid and 1 mL 0.01% (w/v)
bromocresol green aqueous solution were added to 1.0
mL of bisoprolol fumarate solution with a concentration
between 7-80 μg/mL. The complex was then extracted
using chloroform. Ten minutes later the absorbance was
measured at 402 nm, using as reference a blank sample
prepared in the same conditions.
Results and discussions
From the analysis of the absorption spectra (fig. 2), a
maximum absorbance was observed for the reaction
REV. CHIM. (Bucharest) ♦ 65 ♦ No. 8 ♦ 2014 http://www.revistadechimie.ro
Fig. 2. Absorbtion spectra of the
reaction product
Fig. 3. Absorbtion spectra of
bromocresol green (1), bisoprolol (2)
and reaction product (3)
Fig. 4. Absorbtion spectra of
placebo (1), sample solutions (2)
and standard (3)
Fig. 5. Study of stoichiometric ratio
product at 402 nm. This value was used for all the
determinations.
As can be seen in figure 3, bisoprolol and bromocresol
green presented maximum absorbance peaks at 275 nm
and 615 nm, respectively. The specific absorption
coefficient of bisoprolol was A 1%1cm,402nm=250 and the
specific absorption coefficient of the reaction product was
A1%1cm,402nm =960.
Comparing the spectra from figure 4, it was observed
that the proposed method was selective because it had
the ability to separate the corresponding signal of
bisoprolol.
The excipients used in the pharmaceutical formulation
did not interfere with the maximum absorption of
bisoprolol.
The stoichiometric ratio of the reaction product was
investigated by Job’s continuous variation method. For that
study, 1 . 10-2 M bromocresol green and bisoprolol solutions
917

were mixed in varying volume ratio while the total volume
of the mixtures was maintained constant at 10 mL.
From the analysis of figure 5, the optimal combination
ratio of the bisoprolol and bromcresol was established to
be 1:1.
According to table 1, the optimal concentrations of
reagent were 0.1 M for the hydrochloric acid solution and
0.01% (w/v) for the bromocresol green solution.
The stability of the reaction product was evaluated at
room temperature without protection from light. The
absorbance was measured after 10 min extraction in
chloroform according to the experimental data. From figure
6, the absorbance remained almost the same for at least
15 min, time sufficient enough for the analysis to be
performed.
918 http://www.revistadechimie.ro
Table 1
INFLUENCE OF REAGENT
CONCENTRATION
Fig. 6. Stability of the reaction product
Fig. 7. Choice of solvent for the extraction
While choosing the best solvent of the extraction,
experiments were done with ethanol, methanol,
chloroform, diclormethan, acetonitrile (fig. 7). It has been
observed that the best results were obtained when the
extraction was performed in chloroform.
Linearity was assessed by analyzing the obtained data
shown in table 2 by linear regression and the calibration
curve from figure 8 was obtained.
The following calibration curve equation was
established:
Absorbance = 0.012 x Concentration + 0.0034
The parameters obtained for the validation of the
method are summarized in table 3.
Detection (LOD) and quantification limits (LOQ) were
calculated using the following equations [28]:
Table 2
LINEARITY STUDY
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 Fig. 8. Calibration curve

Table 3
VALIDATION PARAMETERS

Table 4
STUDY OF THE PRECISION AND
OF THE ACCURACY OF THE
METHOD

Table 5
ASSAY OF BISOPROLOL IN
PHARMACEUTICAL PRODUCTS

Table 6
COMPARISON WITH OTHER
SPECTROPHOTOMETRIC METHODS

LOD=3.3 x Standard error / Slope = 1.78 μg/mL
LOQ=10 x Standard error / Slope = 5.41 μg/mL
For a 40 μg/mL sample ten determinations were done
under the same experimental conditions for the study of
the suitability of the system. The relative standard deviation
(RSD = 0.67 %) showed that the system was precise.
Three different concentration solutions of bisoprolol
were used thrice for the determination of the precision
and accuracy of the method. The method precision was
investigated through its repeatability and intermediate
REV. CHIM. (Bucharest) ♦ 65 ♦ No. 8 ♦ 2014 http://www.revistadechimie.ro
precision. Standard addition method was used to evaluate
the accuracy of the method. The concentrations of the
samples were calculated using the equation of the
calibration curve (table 4). It was observed that the relative
standard deviation was lower than 2% and thus the method
was precise. Because the recovery was 100.11% it was
concluded that the method was also accurate.
The evaluation of robustness was done by changing the
instrument, and varying the volume of bromocresol green
and the concentration of hydrochloric acid. Reproducibility
of the results confirmed the robustness of the method.
919
 The proposed method has been applied for the analysis
of bisoprolol in tablets. For each sample three
determinations were done. The results listed in table 5
showed that the bisoprolol content (mg/tablet) was ranging
within the limits imposed by 10 th Edition Romanian
Pharmacopoeia – [29]. Excipients were found not to
interfere in the analysis.
The new method was compared with other
spectrophotometric methods. It was observed that the
results were similar to those already published (table 6).
Conclusions
A new spectrophotometric method was developed for
the assay of bisoprolol using bromocresol green as reagent
in acidic medium. The proposed method was based on
the formation of a specific colored product. The reaction
product extracted in chloroform showed a maximum
absorbance peak at 402 nm. The specific absorption
coefficient of bisoprolol and that of the corresponding
reaction product were: and . 17. LI, S., LIU, G., JIA, J., LIU, Y., PAN, C., YU, C., et al. J. Chromatogr.
The analytical method was optimized and validated by
establishing the linearity domain (in the concentration
range 7 - 80 μg/mL), the correlation coefficient (r = 0.9998),
the detection limit (1.78 μg/mL), precision (RSD = 1.06)
and the accuracy (mean recover y = 100.11). The
experimental results concerning the recovery of bisoprolol
in tablets were in accordance with those obtained using
the reference substance.
The main advantage of the new method consists in its
enhanced sensibility, in comparison with other UV
spectrophotometric methods. The new method was
simple and easy to perform and it may be used for quality
control or for routine analysis of pharmaceutical products
when cheap and fast measurements are essential.
Acknowledgements: This research was financially supported by the
European Social Fund within project POSDRU/107/1.5/S/78702.
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Manuscript received: 19.12.2013
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