Monitoring of bisoprolol fumarate stability under different stress conditions
1* 2 2
Irena Kasagiü-Vujanoviü , Biljana Janþiü Stojanoviü , Darko Ivanoviü
*
Corresponding author: kasagic.irena@gmail.com
1
University of Banja Luka–Faculty of Medicine, Department of Drug Analysis, Banja Luka, Bosnia and Herzegovina
2
University of Belgrade–Faculty of Pharmacy, Department of Drug Analysis, Belgrade, Serbia
Abstract. Stability studies of drugs by stress study is a very
important process which is done by treating the study drug
with different stress agents, with the aim to define the critical
factors affecting the stability of the drug, to accurately define
the storage conditions of the drug, as well as to identify the
resulting degradation products. In this paper, stress studies of
bisoprolol fumarate were performed, in order to examine
what are the stress agents that affect its stability.For the anal-
ysis previously optimized and validated HILIC method was
usedIt was demonstrated that oxidative stress agent has the
largest effect on the degradation of bisoprolol fumarate, and
then the acid and base stress agent successively. Water, as a
neutral medium, and light had no significant effect on the
stability of bisoprolol fumarate. During degradation under
acid conditions impurity A was formed and it was confirmed
with UPLC/MS/MS method. In order to more clearly define
the processes of degradation, kinetic studies of degradation of
bisoprolol fumarate have been carried out, in order to deter-
mine the order of the reaction rate of degradation and degra-
dation half-time, which provided clearer definition of the
mechanism of degradation.
Keywords: bisoprolol fumarate, stress study, HILIC, LC-
MS/MS, kinetic studies
Introduction
Forced degradation studies are an important part of the
process of drug development. These studies are conducted
in order to detect the degradation time of the test compound
and defining their stability, as well as to identify potential
degradation products. Information obtained by this way are
used for defining the storage conditions of the drug, as well
as the way of production and drug compatibility with cer-
tain drug excipients [1, 2]. These studies should be conduct-
ed according to the ICH Guideline Q1A(R2) [3]. The exper-
imental conditions for carrying out forced degradation stud-
ies should include testing of drug sensitivity to: hydrolysis,
oxidation, thermal degradation, moisture and light. Forced
degradation studies are carried out on one series of product,
and experimental conditions should be much more extreme
© Springer Nature Singapore Pte Ltd. 2017
A. Badnjevic (ed.), CMBEBIH 2017,
IFMBE Proceedings 62,
DOI: 10.1007/978-981-10-4166-2_64
than in an accelerated stability study. The exact conditions
of performing forced degradation studies are chosen based
on physical and chemical characteristics of the drug [4]. In
this paper, forced degradation studies conducted on
bisporolol fumarate (BF) is presented. Degree of degrada-
tion was followed by previously validated Hydrophilic In-
teraction Liquid Chromatography (HILIC) with UV detec-
tion [5]. In order to confirm structure of main degradation
product UPLC/MS/MS method was used. Literature survey
shown that there are no papers dealing with monitoring of
BF degradation by HILIC method. This is the first time that
degradation of BF was followed by HILIC which is espe-
cially useful when more polar compounds appear in degra-
dation process. Some previously published papers related to
degradation of BF are given in the papers described in the
references 6–9. No data on this subject.
Materials and Methods
Chromatographic system. The analysis was done on the
chromatographic system of Agilent Technologies HP1200,
consisting of HP1200 binary pump, HP1200 UV/VIS
(DAD) detector and ChemStation Software on Windows XP
for data processing.
LC-MS/MS system. Waters ACQUITY UPLC System,
Waters Corporation, USA; Pump: Varian HS 602 vacuum
pump; Autosampler: Acquity FTN; Thermostat: Acquity
Detector: Acquity UV/VIS detector and a mass detector
Tandem Quadrupole Xevo TQ-MS (Waters Corporation,
USA). Desktop Publishing: MassLynx V.4.1 SCN843 soft-
ware, Windows XP; Column: Acquity C18 (100 mm x 2.1
mm, 1.7 microns particle size).
Reagents. For preparation of mobile phase and solution
HPLC-grade reagents were used: acetonitrile (Fisher Scien-
tific, England), concentrated acetic acid (Lachner, Czech
Republic), ammonium acetate (Lachner, Czech Republic)
and HPLC water. For analysis working standards of
415
 4Monitoring of bisoprolol fumarate stability under different stress
H
OH
O H
CH3
O
O CH3
bisoprolol fumarate
Figure 1. The chemical structure of BF
bisoprolol fumarate, impurities of bisoprolol fumarate A, K
and L (Sigma-Aldrich, Germany) were used.
-1
Solutions. The stock solution of BF (c = 1 mg mL ) was
prepared in acetonitrile. Working solutions of BF with con-
-1
centration of 100 ȝg mL was prepared by diluting stock
solution with an appropriate stress agent. As stress agents
were used: 0.01 M and 0.1 M sodium-hydroxide, 0.1M and
0.01M hydrochloric acid, and hydrogen-peroxide in concen-
tration of 3%, 15% and 30%. Also, testing was carried out
under neutral conditions, wherein distilled water was used
as solvent. For degradation performance under the light
influence, during the day natural daylight in combination
with artificial white light was used or only artificial white
light during night, with 12 lamps-set. The strength of one
lamp was 18W (F74-765 daylight – 1,200 lumens,
Tungsram, Hungary). Immediately, following the addition
of stress agents, analysis were carried out and chromato-
grams were developed out of samples. Then, degradation of
all BF samples, was followed after 1h, 24h, 48h and 72h,
without stopping the degradation reaction. All assays were
performed at room temperature (~25°C) analysis in the acid
medium (0.01M HCl), basic medium (0.01M NaOH) and
water were conducted at elevated temperature (50°C).
-1
The stock solutions of impurities (c = 0.1 mg mL ) was
prepared in acetonitrile, and their working solutions of 10
-1
ȝg mL concentration, were prepared by diluting with the
mobile phase.
For MS/MS analysis solution of BF was prepaired in
-1
concentration of 100 ng mL and for impurities in concen-
IFMBE Proceedings Vol.
I. Kasagiü-Vujanoviü et4
N CH3
HOOC
CH3
,
COOH
-1
tration of 10 ng mL . All solutions were prepaired in sol-
vent consisted of 20:80 V/V methanol:water.
Chromatographic conditions (HILIC). Column Luna-5ȝ-
HILIC 200A (100 mm x 4.6 mm, 5 mm particle size), the
mobile phase consisted of a mixture of acetonitrile-water
solution (10 mM ammonium-acetate,pH 4.0, adjusted with
concentrated acetic acid) at a ratio of 92:8 V/V. The mobile
-1
phase flow 1 mL min , column temperature 30°C, detection
wavelength 230 nm and the injection volume of 20 ȝL.
LC-MS/MS conditions. Mobile phase (A): 5 mM aque-
ous solution of ammonium-formate containing 0,5% formic
acid, mobile phase (B): methanol, gradient program: 0 min.
A 85%; 2.5 min. A 60%; 3.9 min. A 5%; 5.2 min. A 60%; 6
min. A 85%; column temperature: 30°C; flow rate: 0.4 mL
-1
min and injection volume: 5 ȝL. Ion sources: ESI, positive
ions analysis (ES+); Mass analyzer: quadrupole; The flow
-1
of drying gas (nitrogen): 12.0 L min ; pressure of gas spray:
60 psi; the temperature of capillary 350°C; voltage of capil-
laries: 3.20 kV; Collision energy: 30 V.
Results and Discussion
Structure of BF is presented in Figure 1.
Taking into account functional groups presented in struc-
ture of BF, it can be expected that BF will be susceptible to
oxidative degradation. But, it is necessary to conduct stress
degradation studies in order to confirme degradation path-
way of BF Forced degradation studies was conducted under
 4Monitoring of bisoprolol fumarate stability under different stress I. Kasagiü-Vujanoviü et4

Table 1. The degree of degradation of BF after the stress study in a defined time interval

The time of deg-

0 1 24 48 72
radation [h]

water 0 0.17 0.18 0.22 0.22

light 0 0.19 0.19 0.24 1.14

The degree of degradation [%]

0.1M
6.89 8.21 11.0 14.98 24.54
NaOH

0.01M
3.01 3.39 3.42 3.86 4.20
HCl

3%
11.17 27.23 33.0 49.30 61.59
H2 O2

15%
49.19 50.44 68.93 75.01 85.11
H2 O2

30%
72.87 100.0 – – –
H2 O2

Figure 2. Chromatogram of BF after treatment with 0.01M NaOH

different stress conditions (see Experimental part) and de- sumed that the BF partially succumbed hydrolysis process.
gree of degradation is given in Table 1. However, there was no formation of degradation products.
Stability tests of BF in neutral medium (water), showed Tests were further conducted at elevated temperature (50°C)
great stability, ie. for 72h < 1% has degraded (Table 1), and in a milder medium (0.01M NaOH), with which was con-
no significant degradation or degradation products for- firmed that degradation degree was much higher (97.28%
mation has happened. Then, tests were conducted at elevat- for 48h in 0,1M NaOH and ~8% for 72h in 0.01M NaOH),
ed temperature, thereby was confirmed that the level of but even in these conditions there has been no occurrence of
degradation was not significantly higher (3.13% for 72h). degradation products (Fig. 2).

In basic medium (0.1M NaOH) at room temperature after Tests carried out in acidic medium of 0.01M HCl showed
72h BF degradation was ~24% (Table 1), what can be as- that BF degradation was ~4% for 72h (Table 1). These

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 4Monitoring of bisoprolol fumarate stability under different stress I. Kasagiü-Vujanoviü et4

o o o
Figure 3. Chromatogram of BF after treatment with 0.01M HCl on 25 C, 50 C and 70 C

Figure 4. Chromatograms of identified resulting degradation product

studies were repeated at elevated temperature in 0.01M Futher on, under the influence of oxidizing agent (3%,
HCl, where there was a significant occurrence of degrada- 15% and 30% hydrogen peroxide) degradation was occur
tion products (Fig.3). Identification was further conducted (Table 1, Fig.6) but obtained degradation products were not
on HILIC method using different standards of impurities detected using applied chromatographic conditions. In order
(A, K and L), where it was confirmed that it was impurity A to get additional information some complementary experi-
(Fig.4). In order to determine with certainty whether it was ments should be conducted.
the impurity, degradation product and then the same sam-
ples were recorded on the mass detector. The obtained MS Stability studies of BF under the influence of light con-
spectra confirmed that the resulting degradation product is firmed that there are no significant degradation (Fig.7).
impurity A (Impurity A: m/z 240,02 ĺ m/z 133,02,
m/z
74,32 and BF: m/z 326,21 ĺ m/z 116,01, m/z 73,99)
(Fig.5).
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 4Monitoring of bisoprolol fumarate stability under different stress I. Kasagiü-Vujanoviü et4

Figure 5. Mass spectrum for the identification of degradation product

Figure 6. Chromatogram of BF after treatment with 15% H2O2

After forced degradation studies, kinetic studies were
carried out in order to define the mechanism of degradation,
predicting rates of degradation reaction, as well as to gain a order of reaction. The rate constant is calculated from the
better insight of BF stability. Based on this analysis it is
possible to make a definitive conclusion about the behavior
and degradation profile of BF. Some important details relat-
ed to kinetic studies are given in references [10, 11]. The
reaction rate is determined by the speed of the reduction of
reactants concentration or by the speed of increasing the
reaction products concentration. For description of reactions
the law of rate of reaction is used. Describing the linear
dependence of changes in BF concentration in precisely
defined time points, correlation coefficient (r) is calculated.
The r-value which shows the highest value for calculated
reaction rate of zero-, first- or second-order, defines the
straight line slope.
Examination of degradation kinetics has shown that the
BF degradation in water is a first-order reaction. The value
-1
of reaction rate constant was 0.0000h , while the reaction
half-time was 0h.
This shows that BF is very stable in the water and during
these tests, degradation has not happened (Table 2, Fig.8).

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 4Monitoring of bisoprolol fumarate stability under different stress I. Kasagiü-Vujanoviü et4

Figure 7. Chromatogram of BF after treatment with light

Table 2. Studies of kinetic degradation of BF in water

time [h] c (concentration) [mM] ln (c) 1/c
0.000 0.129 –2.045 7.728
0.300 0.129 –2.045 7.728
1.000 0.130 –2.039 7.685
24.000 0.130 –2.039 7.680
48.000 0.130 –2.039 7.680
72.000 0.129 –2.047 7.740
slope 0.0000 0.0000 0.0001
intercept r 0.1298 –2.0418 7.7044
0.1008 0.1665 0.1054
Investigating kinetic decomposition showed that base -1 -1
constant was 0.1328 mM h , while reaction half-time was
degradation in 0.1M NaOH for BF is zero-order reaction, 59.17h. BF oxidative degradation in 15% H2O2 is the first-
and r-value for zero-order reaction had the highest value order reaction (Table 6, Fig.12). The rate constant value was
(0.9747), which indicates its highest linearity (Table 3, -1
0.0197h , while reaction half-time was 35.18h. From the
Fig.9). The value of reaction rate constant was expressed as data obtained from degradation kinetics studies under the
the absolute value of the obtained straight line slope which
-1
action of hydrogen-peroxide, it can be seen that of all tested
was 0.0003 mM h , while the reaction half-time was 205.3h stress conditions, this degradation process is the most ex-
calculated from the equation: [A0] - the initial concentration pressed.
of 0.123 mM of BF in 0 minute.
Degradation kinetics examination under the light influ-
The reaction of acid degradation was tested under the in- ence has shown that the BF degradation BF by photolysis is
o
fluence of 0.01M HCl on 25 C. The BF degradation with second-order reaction (Table 7, Fig.13). The reaction rate
acid is first-order reaction. Reaction rate constant value was -1 -1
constant value was 0.0013 mM h , while the reaction half-
-1
0.0013h , while reaction half-time was 533.2h (Table 4, time was 5834.3h. From results we can conclude that BF is
Fig.10). stable to photolysis and requires a long time to degrade
under the influence of light.
Oxidative degradation of the BF with 3% H2O2 is the se-
cond-order reaction (Table 5, Fig.11). The reaction rate

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4Monitoring of bisoprolol fumarate stability under different stress I. Kasagiü-Vujanoviü et4

Figure 8. The graphical determination of the reaction order of BF degradation in water

Table 3. Studies of kinetic degradation BF in 0.1M NaOH

c (concentration) [mM]
time [h] ln (c) 1/c

0.000 0.123 –2.094 8.11

0.300 0.123 –2.099 8.15
1.000 0.122 –2.106 8.21
24.000 0.119 –2.130 8.41
48.000 0.113 –2.184 8.87
72.000 0.101 –2.290 9.87
slope –0.0003 –0.0024 0.021
intercept r 0.1234 –2.0914 8.083
–0.9747 –0.9695 0.963

Figure 9. The graphical determination of the reaction order of BF degradation in 0.1M NaOH

Table 4. Studies of kinetic degradation of BF in 0,01M HCl

c (concentration) [mM]
time [h] ln (c) 1/c

0.000 0.126 –2.072 7.94

0.300 0.124 –2.084 8.03
1.000 0.123 –2.094 8.11
24.000 0.121 –2.096 8.13
48.000 0.120 –2.100 8.16
72.000 0.197 –2.201 9.03
slope 0.0007 –0.0013 0.011
intercept r 0.1173 –2.0758 7.966
0.7355 –0.8542 0.850

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4Monitoring of bisoprolol fumarate stability under different stress I. Kasagiü-Vujanoviü et4

Figure 10. The graphical determination of the order of reaction degradation BF in 0.01M HCl

Table 5. Studies of kinetic degradation of BF in 3% H2O2

c (concentration) [mM]
time [h] ln (c) 1/c

0.000 0.127 –2.063

0.300 0.098 –2.322 10.20
1.000 0.096 –2.345 10.43
24.000 0.090 –2.413 11.16
48.000 0.067 –2.706 1
72.000 0.052 –2.965 19.39
slope –0.0008 –0.0100 0.132
intercept r 0.1074 –2.2263 9.122
0.9099 0.9491 0.963

Figure 11. The graphical determination of the reaction order of BF degradation in 3% H2O2

Table 6. Studies of kinetic degradation of BF in 15% H2O2

time [h] c (concentration) [mM] ln (c) 1/c

0.000 0.074 –2.603 13.49

0.300 0.072 –2.630 13.88
1.000 0.068 –2.686 14.66
24.000 0.040 –3.232 25.33
48.000 0.033 –3.401 29.00
72.000 0.016 –4.136 62.56
slope –0.0008 –0.0197 0
intercept r 0.0695 –2.6377 12
0.9716 0.9843 0

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 4Monitoring of bisoprolol fumarate stability under different stress I. Kasagiü-Vujanoviü et4

Figure 12. The graphical determination of the order of reaction degradation BF in 15% H2O2

Table 7. Studies of BF kinetic degradation under the influence of light

c (concentration) [mM]
time [h] ln (c) 1/c
0.000 0.132 –2.026 7.58
0.300 0.132 –2.026 7.58
1.000 0.132 –2.026 7,58
24.000 0.132 –2.022 7.55
48.000 0.133 –2.018 7.52
72.000 0.134 –2.013 7.48
slope 0.0000 0.0002 –0
intercept r 0.1319 –2.0262 7
0.9969 0.9997 0

Figure 13. The graphical determination of the reaction order of BF degradation under the influence of light

Concentration change with time, provides detailed de- Conclusion

scription of the reaction rate, but it is desirable to have a
simple measure of the reaction rate, and that is exactly the
reaction half-time. The faster the reaction, the shorter the
reaction half-time, so it can be concluded that BF oxidative
degradation is faster reaction in comparison with acid and
alkaline degradation, as well as in comparison with photoly-
sis and degradation in water. This analysis confirmed that
the most pronounced BF degradation is with oxidative deg-
radation, and degradation under the influence of light is
very small.
Description of BF degradation under all tested conditions
represents a significant source of data, which can be very
useful to analyze the impact of external factors on the quali-
ty and effectiveness of this drug which is on the market
mainly in the form of tablets. In order to provide the patient
safe, quality and effective medicine, it was concluded that
the critical factors that influence the stability must be re-
duced to the lowest possible level. Based on detailed analy-
sis, it has proven that the oxidation and acid hydrolysis were
one of the main degradation processes of BF, and that the
temperature is very important factor that accelerates its
degradation. This paper also showed that the chemical ki-
netics application for prediction of pharmaceutical products

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 4Monitoring of bisoprolol fumarate stability under different stress
stability is of great importance and that we can obtain sig-
nificant and reliable data on the stability by defining the
degradation rate of active substance. For stability determi-
nation using this method, the analysis of chemical structure
of explored analytes, the possible route of each compound
degradation can be predicted with great certainty.
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