A HPLC Method for the Determination of Bisoprolol in Tablets

and its Application to a Bioequivalence Study

ALINA-DIANA PANAINTE1, LUMINITA AGOROAEI2*, NELA BIBIRE1, GLADIOLA TANTARU1, MIHAI APOSTU1, MADALINA VIERIU1,
FLORIN ADRIAN SPAC3
1
“Grigore T. Popa“ University of Medicine and Pharmacy, Faculty of Pharmacy, Analytical Chemistry, 15 University Str., 700115,
Iasi, România
2
“Grigore T. Popa“ University of Medicine and Pharmacy, Faculty of Pharmacy, Toxicology, 15 University Str., 700115, Iasi, România
3
“Grigore T. Popa“ University of Medicine and Pharmacy, Faculty of Pharmacy, Physical Chemistry, 15 University Str., 700115, Iasi,
România

A fast, robust RP-HPLC method was developed for determination of bisoprolol fumarate in tablets. The
mobile phase was a mixture of methanol:acetonitrile:45mM potassium dihydrogen phosphate buffer
(30:25:45) at pH 3.0 and 0.3mL/min. The stationary phase used was a SB-C18 Nucleosil column (125×4mm).
The UV detection was performed at 225nm. The method was validated as far as linearity, limit of detection,
limit of quantification, precision, accuracy, recovery and system suitability. The retention time for bisoprolol
was 2.32 min. The calibration graph was linear in the concentration range 0.3-10μg/mL. The assay proved to
be sensitive, specific and reproducible. The method was applied for the determination of bisoprolol in tablets
and in a bioequivalence study.
Keywords: bisoprolol, HPLC method validation, bioequivalence study.

Bisoprolol fumarate is a synthetic beta 1 – selective Experimental part

blocking agent. It is used in the treatment of cardiovascular Instruments
diseases such as high blood pressure (hypertension), An Agilent Technologies 1100 High Performace Liquid
reduced blood flow to the heart (cardiac ischemia) and Chromatograph with Diode Array Detector, a Kern 770
congestive heart failure. It is also used as preventive analytical balance and a Cole Parmer ultrasonic bath were
treatment before and as primary treatment after heart used [13-15].
attacks decreasing the chances of recurrence [1]. The chromatographic separation of bisoprolol was
Bisoprolol has a higher degree of beta 1 - selectivity achieved using isocratic elution and a reverse phase
compared to other beta 1 - selective beta – blockers such column (Zorbax SB-C18 100×3mm, 3.5μm). The
as atenolol, metoprolol and betaxolol) [2-4]. The beta 1 – chromatographic assay was performed at 25°C using a
selectivity of bisoprolol fumarate has been demonstrated 20μL sample loop.
in both animal and human studies.
The chemical name for bisoprolol fumarate is (±)-1-[4- Reagents
[[2-(1-methylethoxy) ethoxy] methyl]phenoxyl-3-[(1- The solvents and other materials were HPLC grade
methylethyl)amino]-2-propanol(E)-2-butenedioate [5]. Its provided by Merck’s Chemical Co and Fluka. The bisoprolol
empirical formula is (C18H31NO4)2·C4H4O4 and its structure reference substance was supplied from Unichem
is presented in figure 1: Laboratories LTD, India. Pharmaceutical formulations of 5
and 10mg bisoprolol tablets were acquired from local
CH3 pharmacies.
Fig. 1. Structural
O N
H
CH3 formula of bisoprolol Mobile Phase
OH fumarate The mobile phase consisted from a 30:25:45 mixture of
CH COOH methanol, acetonitrile and 45mM potassium dihydrogen
CH COOH pH 3.0 phosphate buffer and it was pumped into the system
with a flow rate of 0.30mL/min.
O CH3
O
Preparation of solutions
CH3 2 100 μg/mL stock standard solution of bisoprolol was
Various methods for bisoprolol determination are prepared using mobile phase and then stored at room
reported in literature. There are UV-Vis [6, 7], HPLC [8 – temperature. The stock standard solution was diluted with
10], HPTLC [11, 12] methods for the determination of mobile phase to appropriate working solution (10μg/mL).
bisoprolol from tablets and biological fluids. A phosphate buffer (45mM) was prepared by dissolving
The aim of this study was to develop a fast, efficient, 6.12g of monopotassium phosphate in 900mL water. The
accurate, precise and robust RP-HPLC method for the solution was brought to 1000mL with distilled water and
separation and quantitative determination of bisoprolol from the pH was adjusted to 3.0 with ortophosphoric acid.
tablets.
* email: [email protected]

REV. CHIM. (Bucharest) ♦ 66 ♦ No. 11 ♦ 2015 http://www.revistadechimie.ro 1791

Sample preparation was establishing the composition of the mobile phase and
The developed method for the determination of the retention time (fig. 2). According to the obtained results
bisoprolol was applied to a quality control study of it was established that the best mobile phase was a
pharmaceutical tablets from various manufacturers. mixture of acetonitrile, methanol and pH 3.0 phosphate
Twenty bisoprolol fumarate tablets were crushed and a buffer and the retention time of bisoprolol fumarate was
5mg bisoprolol fumarate equivalent quantity of powder 2.32min.
was dissolved in 25mL methanol using an ultrasonic bath
and then it was filtered in order to separate the excipients.
It was all brought to 100mL using mobile phase. Three
successive samples were obtained.
The bioequivalence study
The pharmaceutical monography for tablets provides a
quantitative determination of the active substance released
after tablet dissolution. The tablets analysed were Concor®
(original product) and Bisotens® (generic product), both
containing 10mg bisoprolol fumarate. The dissolution test
was carried out on 6 tablets, using phosphate buffer as
dissolution medium, according to United States
Pharmacopeea 29 (USP) [5], at three different pH values
(1.2, 4.5 and 6.8) using an Agilent Technologies 708
dissolution apparatus. The dissolution mediums were Fig. 2.Retention time
prepared according to European Pharmacopoeia 6.0 [13-
15].
Method validation
Validation of the method was carried out following the
norms of The International Conference on Harmonization
(ICH) [16] guidelines for selectivity, linearity, detection limit,
quantification limit, system suitability, precision, accuracy,
recovery, stability and robustness.
The linearity was investigated in the 0.10-15μg/mL
concentration range and the calibration curve was
obtained by plotting the peak area values against the
bisoprolol fumarate concentration using linear regression.
The detection limit (LOD) and the quantification limit
(LOQ) were calculated using the calibration curve
statistical parameters in the following formulas (1): Fig.3. The flow rate study

(1)

where S was the slope of the calibration curve and SD was

the standard deviation of the intercept of from regression
equation [16-19].
The precision and accuracy were assessed by
determining the active compound concentration at three
concentration levels in the same day and in three different
days. The precision of the method was evaluated through
standard deviation (SD) and covariance (CV) (%),
respectively. The accuracy was evaluated through recovery.
The recovery of tablet extraction for three bisoprolol
fumarate concentrations was determined. Known
amounts of standard bisoprolol fumarate were added to
tablets solutions. After the chromatographic analysis, the Fig.4. The absorbtion spectra of bisoprolol fumarate
peak areas were compared to those obtained for standard
solutions of bisoprolol fumarate with equal concentration. Various proportions of acetonitrile, methanol and
To evaluate the robustness of the developed RP-HPLC phosphate buffer were tested when establishing the
method, some small deliberate variations in the optimized optimum mobile phase: 20:30:50, 25:30:45, 30:25:45 and
method parameters were made. The effects of modifying 30:20:50. The most appropriate chromatographic peaks
flow rate or column temperature on the peak area were were obtained with a 25:30:45 (v/v/v) mobile phase. Then
studied. the influence of the mobile phase flow rate on peak
A series of standards samples was prepared from fresh normalization was studied (fig. 3).
stock solutions in the mobile phase to evaluate the stability The optimum flow rate was 0.3mL/min. The detection
of samples at room temperature. was performed at a wavelength of 225nm as it was
determined from the absorption spectra of bisoprolol
Results and discussions fumarate (fig. 4).
Optimization of chromatographic conditions The pH effect on the peak area of a 5μg/mL bisoprolol
Several parameters were examined for the optimization fumarate sample was examined. Figure 5 shows the pH
of HPLC analysis of the bisoprolol fumarate. The first step variation.
1792 http://www.revistadechimie.ro REV. CHIM. (Bucharest) ♦ 66 ♦ No. 11 ♦ 2015
 Fig. 5. The pH effect

Fig. 6. Chromatograms for blank

(0.10%), standard (9μg/mL), and
sample (0.60μg/mL)

Table 1
SYSTEM SUITABILITY

Method validation The system suitability was evaluated using bisoprolol

The selectivity was assessed by comparing the fumarate standard solution. Then the standard error (SD),
chromatograms for standard, sample and blank solutions theoretical plate number (N), capacity factor (k‘) and tailing
(fig. 6). factor (T) were determined. The results were all within
That figure showed that the method was selective acceptable limits (table 1).
because it had the ability to separate the signal Precision and accuracy were tested for three different
corresponding to bisoprolol. Also, the excipients used in concentrations (3, 5 and 7μg/mL). Table 2 summarizes
pharmaceutical formulations had retention times that did the results obtained for the intraday parameters. The
not interfere with the retention time of bisoprolol. interday precision and accuracy were evaluated for six
For linearity study a calibration graph (fig. 7) plotted the aliquots of three sample concentration in three different
concentration against bisoprolol fumarate peak area; that days. The results are presented in table 3. The intra and
graph showed a good linearity in 0.30-10μg/mL interday precision of measurements were lower than the
concentration range. Correlation coefficient was 0.9995. accepted criteria (CV ≤15%) and concerning the accuracy
The LOD and LOQ of bisoprolol fumarate were 0.10 and of the method, the recovery variation was in between 99.33
0.28µg/mL, respectively. Those values were lower than and 102.00%.
those obtained in many other reported methods.
REV. CHIM. (Bucharest) ♦ 66 ♦ No. 11 ♦ 2015 http://www.revistadechimie.ro 1793
 Fig 7. Calibration curve

Table 2
INTRADAY PRECISION AND
ACCURACY

Table 3
INTERDAY PRECISSION AND
ACCURACY

Table 4
RECOVERY RESULTS OF BISOPROLOL FUMARATE TABLET ASSAY

Table 5
ROBUSTNESS STUDY

Recovery and RSD% values (table 4) obtained during summarised in table 6 and showed that bisoprolol is found
bisoprolol fumarate tablets assay showed that the method to be stable in solution for 8 h at room temperature (CV%
could be applied with good results in drug control analysis. ≤15%).
To test bisoprolol stability in solutions, a number of 6 Analysing the dissolution profiles from figure 8 it was
replicates with 5μg/mL concentration of the quality control observed that the dissolution percentage of both types of
were used and compared with 6 replicates of stability tablets in the specified conditions of USP 29 and at three
samples with the same concentration. The solutions were pH values were higher than 85% (as a percentage of the
kept at room temperature for 8 h. The obtained results are amount claimed). The average amount of bisoprolol
1794 http://www.revistadechimie.ro REV. CHIM. (Bucharest) ♦ 66 ♦ No. 11 ♦ 2015
 Table 6
ANALYTE STABILITY IN SOLUTION AT ROOM
TEMPERATURE

®
Fig 8. Dissolution profiles for Concor (1) and
Bisotens® (2)

fumarate dissolved within 15 min was higher than 95% for 7. ATULA, S., RAJESH, T., SANJAY, SJ. Pak. J. Pharm. Sci., 21, 2008,
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8. HEFNAWY, MM., MAHA SULTAN, MAA., MOHAMED AL-SHEHRI., MM.
Conclusions Chem. Pharm. Bull., 55, nr.2, 2007, p. 227.
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