Polymorphic forms of bisoprolol fumarate: Preparation and characterization

Article abstract of DOI:10.1007/s10973-018-7553-8

Bisoprolol fumarate is a beta blocker-type drug substance which has been well known for several decades. However, no relevant data can be found in the literature about its crystal polymorphism. The purpose of this paper was to present two anhydrous forms (Form I and Form II) and a hydrate of bisoprolol fumarate substance. Crystalline forms were studied by various solid-state analytical methods: Fourier transform infrared (FT-IR) spectroscopy, X-ray powder diffraction (XRPD), dynamic vapor sorption (DVS) and thermoanalytical methods (thermogravimetry and differential scanning calorimetry). Thermodynamic stability and solubility of the presented polymorphs were also investigated. Both FT-IR and XRPD methods were found to be suitable for the characterization of the different crystal structures. Thermoanalytical measurements showed that (1) Form I and Form II own clearly different melting points and (2) both Form II and hydrate forms can transform into Form I at higher temperature values. Results of the DVS measurements prove that both Form I and Form II became metastable under extremely humid conditions (> 80% RH) and converted into the hydrate. Thermodynamic stability studies showed that Form I and Form II polymorphs are in enantiotropic relationship with an enantiotropic point at about 40–45?°C. Solubility studies indicated that all of the prepared forms are highly soluble, and no difference was found between them. Considering the recommendations of the corresponding International Conference of Harmonization guideline, it can be stated that no specification is required for crystal polymorphism in case of this substance.

Full text of DOI:10.1007/s10973-018-7553-8

Journal of Thermal Analysis and Calorimetry
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Polymorphic forms of bisoprolol fumarate
Preparation and characterization
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3
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Kata Judit Do¨mo¨to¨r1,2 Miklos Tamas Katona Imre Markovits2 Judit Vargane Lang
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Adam Detrich
Received: 22 February 2018 / Accepted: 12 July 2018
´
´
Ó Akademiai Kiado, Budapest, Hungary 2018
Abstract
Bisoprolol fumarate is a beta blocker-type drug substance which has been well known for several decades. However, no
relevant data can be found in the literature about its crystal polymorphism. The purpose of this paper was to present two
anhydrous forms (Form I and Form II) and a hydrate of bisoprolol fumarate substance. Crystalline forms were studied by
various solid-state analytical methods: Fourier transform infrared (FT-IR) spectroscopy, X-ray powder diffraction (XRPD),
dynamic vapor sorption (DVS) and thermoanalytical methods (thermogravimetry and differential scanning calorimetry).
Thermodynamic stability and solubility of the presented polymorphs were also investigated. Both FT-IR and XRPD
methods were found to be suitable for the characterization of the different crystal structures. Thermoanalytical mea-
surements showed that (1) Form I and Form II own clearly different melting points and (2) both Form II and hydrate forms
can transform into Form I at higher temperature values. Results of the DVS measurements prove that both Form I and Form
II became metastable under extremely humid conditions ([ 80% RH) and converted into the hydrate. Thermodynamic
stability studies showed that Form I and Form II polymorphs are in enantiotropic relationship with an enantiotropic point at
about 40–45 °C. Solubility studies indicated that all of the prepared forms are highly soluble, and no difference was found
between them. Considering the recommendations of the corresponding International Conference of Harmonization
guideline, it can be stated that no specification is required for crystal polymorphism in case of this substance.
Keywords Bisoprolol fumarate Á Crystal polymorphism Á Thermodynamic stability Á Solubility Á Solid-state analysis
Introduction
substance own exactly the same chemical structure, their
physical and physicochemical properties (e.g., melting
point, solubility, hygroscopic property, thermodynamic
stability, crystallinity) can be significantly different due to
their various crystal structures.
Crystal polymorphism has become an important issue for
the last several decades in the pharmaceutical industry
[1–11]. Though different polymorphic forms of a drug
One of the most relevant matters is the solubility dif-
ference between polymorphs of active pharmaceutical
ingredients (API) [12]. The solubility of the API directly
influences the bioavailability of the final product; therefore,
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s10973-018-7553-8) contains
supplementary material, which is available to authorized
users.
3
´
´
& Adam Detrich
detrich.adam.dezso@egis.hu
Laboratory of Finished Product Analytical Development 3,
Finished Product Analytical Development Division,
Directorate of Finished Product Development, Egis
1
Laboratory of Polymorphism Research, Finished Product
Analytical Development Division, Directorate of Finished
´
Product Development, Egis Pharmaceuticals PLC, Kereszturi
´
´
Pharmaceuticals PLC, Bokenyfoldi ut 116-120,
¨
¨
Budapest 1165, Hungary
4
Technical Purchasing Division, IT and Instrument
Procurement Department, Technical Directorate, Egis
´ ´
Pharmaceuticals PLC, Kereszturi ut 30-38, Budapest 1106,
´
ut 30-38, Budapest 1106, Hungary
2
Drug Substance Pilot Plant, Drug Substance Development
Division, Directorate of Drug Substance Development, Egis
´ ´
Pharmaceuticals PLC, Kereszturi ut 30-38, Budapest 1106,
Hungary
Hungary
123

Á. Detrich et al.
this parameter has a strong effect on its performance. In
case of substances with low solubility, this factor is more
emphasized. In the biopharmaceutics classification system
(BCS), drug substances are categorized based on their
aqueous solubility and permeability (Class I: high solu-
bility, high permeability; Class II: low solubility, high
permeability; Class III: high solubility, low permeability;
Class IV: low solubility, low permeability) [13–15].
The thermodynamic stability is also a very important
aspect of the investigation of different polymorphic forms
[16–18]. In case of drug substances, the risk of polymor-
phic transformation during the formulation process or
storing is increased by choosing a metastable form for drug
product development. Such transformations can threaten
the uniform quality and bioavailability of the final product.
However, a metastable polymorph can be also suitable for
further development if its potential polymorphic transfor-
mations are hindered by choosing the appropriate manu-
facturing and formulation processes and package systems
for the storing of the final product. From these reasons, it is
very important to analyze the thermodynamic stability of
the different polymorphs and the potential transformations
between them [19, 20].
diseases (e.g., high blood pressure, cardiac ischemia, heart
failure). It was patented in 1976 [28], and its fumarate salt
(see Fig. 1) has been used in many various drug products
[e.g., Concor (Merck); Zebeta (Teva); Bisoprolol-ratio-
pharm (Teva); Bisoblock (Actavis); Bisoprolol Sandoz
(Sandoz)]. Although the substance has been known for
such a long time, no comprehensive report can be found in
the literature about its crystal polymorphism.
A monograph about the bisoprolol fumarate substance is
present in both the European (Ph. Eur.) and the American
(USP) Pharmacopoeia [29, 30]. It is mentioned in the Ph.
Eur. monograph that the API shows polymorphism and is
very soluble in water.
Solubility of the substance was investigated in some
studies, and it was classified as a BCS Class I material
[31–33]. Solubility of different polymorphic forms was not
mentioned in these articles. Moreover, it was stated in one
of these papers published in 2014 that ‘‘references to
polymorphic forms were not found in the literature’’ [33].
Bisoprolol fumarate has been used for drug product
development purposes at Egis Pharmaceuticals PLC for
many years. During the study of the API, it was found out
that two different polymorphic forms and a hydrate of the
substance exist which have not been published in the lit-
erature yet. The purpose of this paper was to present the
detailed solid-state analytical investigation of these crys-
talline forms. The thermodynamic stability relationship
between them and their solubility were also studied. Based
on the gathered information, it can be decided whether it is
necessary to specify the crystal polymorphism of bisoprolol
fumarate substance to provide a drug product with
acceptable properties and consistent bioavailability.
Crystalline polymorphic forms of a given substance can
show significantly different behavior under humid condi-
tions [21]. Hygroscopic property can result in undesired
and uncontrolled water uptake which may cause problems
in stability, water content and content uniformity of the
drug product. Polymorphic transformations can also occur
due to the water uptake (e.g., formation of a hydrate
structure from an anhydrous form) [22]. Therefore, this
property has to be carefully studied and considered before
choosing the appropriate polymorphic form for further
development and determining the suitable storing condi-
tions and package systems.
Experimental
Among many others, these reasons indicate that prop-
erties of different polymorphic and pseudopolymorphic
(e.g., hydrates, solvates) forms of the actual substance have
to be studied before drug product development [23–25]. In
accordance with this statement, the guideline prepared by
the International Conference of Harmonization (ICH) rec-
ommends a polymorphism screen on the drug substance
and detailed characterization of the found polymorphs
before use [26, 27]. If only one crystal structure of the
substance is known or no significant differences were
found between the properties of the discovered polymorphs
or the detected differences do not affect the safety, per-
formance or efficacy of drug product, no acceptance cri-
terion is needed for crystal polymorphism according to the
decision tree #4 presented in the ICH guideline [26].
Preparation of the samples
Two different polymorphic forms (Form I and Form II) and
a pseudopolymorphic (hydrate) form of bisoprolol fuma-
rate were prepared. Active substance (bisoprolol or its
fumarate salt) used for the experiments was produced at
Egis Pharmaceuticals PLC. Details of the synthesis and
supplier of the starting materials are confidential informa-
tion. The original form of the substance was Form I, and in
our experience it is valid for most of the commercially
available products. Preparation processes were carried out
as follows.
Form I
Bisoprolol
(1-(propan-2-ylamino)-3-[4-(2-propan-2-
beta
yloxyethoxymethyl)phenoxy]propan-2-ol) is
a
Two different processes were applied for the preparation of
the Form I polymorphic form.
blocker-type drug substance most commonly used for heart
123

Polymorphic forms of bisoprolol fumarate: preparation and characterization
Fig. 1 Chemical structure of
bisoprolol hemifumarate drug
substance
OH
H
OH
H
N
CH
O
O
3
CH
3
CH
3
O
O
HO
O
CH
3
2
and enantiomer
At the first, one bisoprolol base was used as starting
material. In this case, 25.0 g (77 mmol) of bisoprolol base
was dissolved in 70 mL of acetone (for synthesis) in an
EasyMaxTM 102-type reactor system (Mettler Toledo AG)
equipped with 100 mL glass reactors, overhead driven
anchor stirrers, turbidity probes and temperature sensors.
The solution was heated to 40 °C, and 4.45 g (38 mmol) of
fumaric acid (for synthesis) was added. While the suspension
was heated to reflux, it transformed into a clear solution.
After 15 min of reflux, the hot solution was cooled down to
0–5 °C in 2 h: the crystallization of Form I started during the
cooling process. After 1 h of crystallization at 2 °C, the
suspension was filtered, and the crystalline product was
washed with acetone and dried at 50 °C. 25.2 g (85%) of
Form I-type bisoprolol fumarate was produced.
reflux in an EasyMaxTM 102-type reactor system (Mettler
Toledo AG) equipped with 100 mL glass reactors, overhead
driven anchor stirrers, turbidity probes and temperature
sensors. After 15 min of reflux, the hot solution was cooled
down to 0–5 °C in 2 h. At 50 °C, the solution was seeded
with 0.35 g of bisoprolol fumarate (Form II or the mixture of
Form I and Form II forms). After 1 h of crystallization at
2 °C, the suspension was filtered, and the crystalline product
was washed with acetone and dried at 50 °C. 28.55 g (95%)
of Form II-type bisoprolol fumarate was produced.
It should be noted that for the second process previous
preparation of seeding crystals is necessary (via slurry
stirring).
Hydrate
The other preparation process started directly from biso-
prolol fumarate. 30.0 g anhydrous form of the substance was
dissolved in 71 mL of acetone at reflux in the same type
reactor mentioned before. The following steps were exactly
the same as it was presented at the previous process: after
15 min of reflux the hot solution was cooled down to 0–5 °C
in 2 h, the crystallization started, and after 1 h of crystal-
lization at 2 °C the suspension was filtered. The crystalline
product was washed with acetone and dried at 50 °C. 25.0 g
(83%) of Form I-type bisoprolol fumarate was produced.
Purity of the prepared active pharmaceutical ingredient
was found to be 99.88% based on high-performance liquid
chromatography (HPLC) results.
Hydrate form of bisoprolol fumarate substance was pro-
duced by storing either of the anhydrous forms (Form I or
Form II) under humid conditions. For this purpose, 3.3 g of
potassium nitrate (KNO3, for analysis, C 99.0%, Merck)
was dissolved in 10 mL of water in order to get a saturated
solution which was put into a desiccator. A small addi-
tional amount of KNO3 was given to the system (so the
solution contained solid excess of it) to guarantee its sat-
urated state. The equilibrium relative humidity over this
solution is about 93–94% at room temperature [28]. A
small amount of anhydrous bisoprolol fumarate was put
into this desiccator and after at least 24 h of storing the
substance transformed into the hydrate form. After
removing it from the desiccator, it remained stable under
normal conditions (room temperature, * 30–60% RH).
Form II
Form II crystalline form of bisoprolol fumarate can be
produced via slurry stirring or a seeding process.
In the first case, 30.0 g of bisoprolol fumarate (Form I)
was suspended in 70 mL of acetone and stirred for a longer
period at ambient temperature in a round bottom flask
equipped with a magnetic stirrer. After at least 96 h of
stirring, Form I form transformed into Form II polymorph.
Afterwards the suspension was filtered and the yield was
found to be 29.5 g (98%).
Characterization of the samples
Bisoprolol fumarate API samples were characterized by
various solid-state analytical methods.
X-ray powder diffraction (XRPD)
XRPD patterns were obtained using a PANalytical
Empyrean diffractometer. X-ray radiation was produced by
˚
a copper X-ray tube with a wavelength of 1.541874 A (Cu
For the seeding process, 30.0 g of anhydrous form of
bisoprolol fumarate was dissolved in 71 mL of acetone at
123

Á. Detrich et al.
Ka) and was focused by a focusing elliptical mirror.
Accelerating voltage and anode heating current values
were set to 45 kV and 40 mA, respectively. Silicon powder
was used as line position and line shape standard, and
alumina plate was applied as relative intensity standard
(both standards are certified, originated from National
Institute of Standards and Technology, NIST). The
instrument was used in transmission mode, and the powder
samples were placed into the sample holder between two
Mylar foils without grinding. Samples were rotated (1 rps)
during the measurement. A PIXcel 3D 1 9 1 area detector
in scanning line detector (1D) mode was used at the dif-
fracted side. Measurements were taken with a step size of
0.01 °2h, a measurement range of 2.00–35.00 °2h and a
time per step value of about 110 s. The whole process was
carried out at room temperature.
indium standard (NIST). 2–3 mg of powder samples was
used for each measurement. Measurements were taken
using TA Instruments standard aluminum pans (open pans
for hydrate samples and sealed ones for the others,
respectively). Samples were heated up to 120 °C with a
heating rate of 10 °C min-1. Form II samples were ana-
lyzed with moderate heating rate values, too (2; 1 and
0.5 °C min-1, respectively; see ‘‘Thermodynamic stability
of bisoprolol fumarate polymorphs’’ section for details).
For the determination of melting points, three parallel
measurements were performed.
Dynamic vapor sorption (DVS)
DVS measurements were taken using a TA Instruments
Q5000 SA sorption analyzer. Mass was calibrated using a
certified calibration weight (Mettler Toledo AG). Relative
humidity (RH) was calibrated applying salts with different
relative humidity values over their saturated aqueous
solutions. The used salts (and the corresponding RH values
at room temperature based on literature data [34]) were the
following: lithium chloride (LiCl, for analysis, Merck, RH:
11.3%), magnesium chloride hexahydrate (MgCl2*6H2O,
for analysis, Merck, RH: 32.8%), sodium bromide (NaBr,
TA Instruments, RH: 57.6%), sodium chloride (NaCl, for
analysis, Merck, RH: 75.3%), potassium chloride (KCl,
puriss, Sigma-Aldrich, RH: 84.2%) and potassium nitrate
(KNO3, for analysis, Merck, RH: 93.6%). Measurements
were started at the actual relative humidity (40%), and
relative humidity was changed within a cycle as follows:
40%?0%?95%?40%, i.e., a cycle contained a desorp-
tion (40%?0% RH), a sorption (0%?95% RH) and a
second desorption (95%?40% RH) stage. Two cycles
were taken for all studied samples. Step size was 5%, and
the instrument stepped to the next stage if equilibrium state
occurs (or the maximum stage time, set to 360 min
expires). Minimum stage time was set to 5 min, and the
state was considered as equilibrium if the rate of change in
mass per time unit (dm/dt) was below 0.01%. Measure-
ments were taken at 25 °C under nitrogen gas flow (200
mL min-1).
Fourier transform infrared (FT-IR) spectroscopy
FT-IR spectra were taken using a Bruker Alpha FT-IR
spectrophotometer in attenuated total reflection (ATR)
measuring mode. Instrument was calibrated using a poly-
styrene film (certified Mid Infrared Transmittance Wave-
length Standard, Bruker Optik GmbH). Measurements
were taken in the range of 4000–525 cm-1 with a resolu-
tion of 2 cm-1. The number of scans was 16 for each
sample.
Thermogravimetric analysis (TG)
TG measurements were taken using a TA Instruments
Discovery Thermogravimetric Analyzer under nitrogen gas
flow (25 mL min-1). Measured mass and temperature were
calibrated using TGA calibration weights and certified
alumel and nickel Curie standards, respectively (both from
TA Instruments). About 12–15 mg of powder samples was
put into 100 ll platinum pans and was heated up to 120 °C
with a heating rate of 10 °C min-1. Additionally two dif-
ferent sorts of isothermal heat treatment were carried out
for hydrate samples: in the first process sample was heated
up to 80 °C (heating rate: 10 °C min-1) and held at this
temperature for 15 min. In the second one, it was heated up
to 50 °C (heating rate: 10 °C min-1) and held for 60 min
at this temperature value (see ‘‘Thermogravimetric analysis
(TG)’’ section for details).
Investigation of the thermodynamic stability
Thermodynamic stability of bisoprolol fumarate poly-
morphs was investigated via the competitive slurry exper-
iment [35]. In this process, the powder mixture of the
studied polymorphs is suspended in different solvents and
stirred for a longer period. Dynamic dissolution–recrys-
tallization processes take place during the stirring proce-
dure. Since polymorphs have different solubility in general,
the composition of the mixture will change during the
process via solvent mediated polymorphic transformations
Differential scanning calorimetry (DSC)
DSC curves were obtained using a TA Instruments Dis-
covery Differential Scanning Calorimeter under nitrogen
gas flow (50 mL min-1). The instrument was equipped
with an RCS90 Refrigerated Cooling System. Measured
temperature and enthalpy were calibrated using certified
123

Polymorphic forms of bisoprolol fumarate: preparation and characterization
6.80 0.05 using 0.1 M NaOH solution. Finally, it
was filled up to volume with water and mixed.
and the mixture will be enriched with the polymorph with
lower solubility (in the actual solvent). If we keep on
stirring the suspension for a sufficient time, only one
polymorph will remain in the sample (which has the worst
solubility in the actually used solvent). Since the thermo-
dynamically stable form owns worse solubility compared
to the metastable ones, the stable polymorph can be iden-
tified at the end of this experiment by filtering the sub-
stance from the suspension and taking its X-ray powder
diffractogram. It is important to mention that this method
does not work if a hydrate or a solvate forms during the
stirring process. In this case, we are not able to provide any
information about the thermodynamic stability of the
starting polymorphs. From this reason, experiment should
be repeated using different solvents with various charac-
teristics. In this case, the 1:1 mixture of Form I and For-
m II forms of the substance was suspended in six different
solvents (ethyl-acetate, isopropanol, tetrahydrofuran, etha-
nol, diethyl-ketone and acetone) and stirred for 1 day at
room temperature (25 °C). All of the used solvents were
synthesis grade and manufactured by pharmaceutical raw
material producers. Filtered samples were studied by
XRPD.
The equilibrium solubility values for bisoprolol fuma-
rate substance were measured by the saturation shake-flask
method [36] using temperature-controlled orbital agitation
platform (GFL 1029 Shaking Water Bath).
For carrying out a measurement, 10 mg of the actually
studied bisoprolol fumarate polymorph was weighed into a
25-mL Erlenmeyer flask and then 10.0 mL of the appro-
priate medium was added. The solution was stirred for a
period of 6 h (saturation time) at controlled temperature
(37.0 0.1 °C) for achieving the thermodynamic equi-
librium. After a further 18 h of sedimentation at the same
temperature value (to achieve the separation of the excess
solid from the solution), samples were filtered and ten
times diluted solutions were studied applying a suit-
able HPLC method described in ‘‘High-performance liquid
chromatography (HPLC)’’ section. Three parallel solubility
experiments were performed in each medium.
High-performance liquid chromatography (HPLC)
Amount of dissolved bisoprolol fumarate substance in the
solutions prepared for solubility studies (see ‘‘Solubility
studies’’ section) was measured by HPLC. For the mea-
surements, an Agilent 1200 Series instrument was used
equipped with a UV detector (operating at a wavelength
value of 226 nm). The used column was Nucleosil C8,
5 lm, CC 250 mm 9 4 mm (Macherey–Nagel). Mea-
surements were taken at 60 °C and 85 bar, and the flow
rate was set to 1.0 mL min-1. 250:750 (V/V) mixture of
acetonitrile and triethylamine (TEA) buffer (pH = 3.0) was
used as eluent applying isocratic elution. TEA buffer was
prepared by adding 7.0 mL of TEA (for synthesis) to about
980 mL of water (HPLC grade). pH of the buffer was
adjusted to 3.0 using concentrated phosphoric acid (H3PO4,
excipient grade), and finally, it was filled up to 1000 mL
with water. The injected sample volume was 20 lL, and
samples were stored at 25 5 °C before the measurement.
Time of the measurement was 16 min, as the retention time
for the bisoprolol substance was found to be about 5.4 min.
Dissolved amount of bisoprolol was determined based on
the area under the appropriate peak and using external
standard calibration.
Solubility studies
Solubility of three different bisoprolol fumarate poly-
morphs or pseudopolymorphs (Form I, Form II and
hydrate) was tested in three different dissolution media
such as 0.1 M HCl solution, pH = 4.5 phosphate buffer and
pH = 6.8 phosphate buffer. Dissolution media were pre-
pared as follows:
1. 0.1 M hydrochloric acid solution About 750 mL of
water was added into a 1000-mL volumetric flask, and
then 8.4 mL of 37% hydrochloric acid (excipient
grade, 37%, Merck) was weighed accurately into the
flask. Finally, it was filled up to volume with purified
water and mixed.
2. pH = 4.5 phosphate buffer solution 13.61 g of potas-
sium dihydrogen phosphate (KH2PO4, excipient grade,
Thomasker) was accurately weighed into a 1000 mL
volumetric flask and dissolved in 750 mL of purified
water. The pH of the solution was adjusted to
4.50 0.05 using 0.2 N phosphoric acid (excipient
grade, 85%, Merck) if it was necessary. Then, the
solution was filled up to volume with water and mixed.
3. pH = 6.8 phosphate buffer solution 6.81 g of KH2PO4
and 0.9 g of sodium hydroxide (NaOH, excipient
grade, Molar) were accurately weighed into a 1000-mL
volumetric flask and dissolved in 750 mL of purified
water. The pH of the solution was adjusted to
Results and discussion
Characterization of crystal structure
Polymorphic forms of bisoprolol fumarate substance were
investigated by XRPD and FT-IR spectroscopy.
123

Á. Detrich et al.
XRPD results showed that all of the studied forms are
crystalline, diffractograms contained sharp and well-de-
fined peaks (see Fig. 2). Diffractograms of the three pre-
sented forms are clearly different, and they can be
unambiguously distinguished.
90
70
Form I
Form II
50
100
80
FT-IR spectra taken from the three forms of bisoprolol
fumarate are plotted in Fig. 3. FT-IR is a suitable method
for the investigation of the chemical structure of the
molecule, since characteristic bands and the corresponding
wavenumber values are typical for the appropriate func-
tional groups. Carrying out the measurements in solid-state
FT-IR can be also suitable for distinguishing different
polymorphic forms of substances with the same chemical
structure.
60
100
Hydrate
80
60
3800 3200 2600 2000 1700 1400 1100
Wavenumber/cm–1
800 600
Fig. 3 Fourier transform infrared spectra for polymorphic (Form I
and Form II) and pseudopolymorphic (hydrate) forms of bisoprolol
fumarate substance
FT-IR spectra were evaluated as characteristic vibra-
tions, and functional groups are given in Table 1 together
with the corresponding wavenumber values [37]. There are
slight differences between the corresponding wavenumber
values for each polymorphic form depending on the actual
crystal structure. The vibration belonging to the crystalline
water obviously appears only in the spectrum of the
hydrate form (first line in Table 1).
solvate free (anhydrous); they do not contain any volatile
components.
For the hydrate form, a mass loss of 2.0% was detected
between 30 and 120 °C which is probably related to the
evaporation of its water content. The measured value is in
reasonable agreement to the water content of a monohy-
drate (molar ratios: 2:1:1 for bisoprolol base, fumaric acid
and water, respectively, calculated water content: 2.3%).
Derivative TG (DTG) curve for hydrate was also plotted. It
clearly showed that dehydration underwent via more than
one steps. The process can be divided into three different
stages with the corresponding temperature ranges of 25–75,
75–90 and 90–120 °C, respectively.
Both XRPD and FT-IR methods are suitable for the
characterization of the crystal structure of the substance in
solid state.
Thermoanalytical investigations
Thermogravimetric analysis (TG)
Water was evaporated from the studied sample by
isothermal heat treatment, too, carried out in the TG
instrument. Two different processes were applied:
Bisoprolol fumarate samples of Form I, Form II and
hydrate were analyzed applying the TG method (see
Fig. 4). Samples were heated up to 120 °C with a heating
rate of 10 °C min-1
.
1. In the first case, sample was heated up to 80 °C and
held at this temperature for 15 min. After all the
volatile compounds were removed from the sample
(the mass change was found to be 1.9%), it was studied
by XRPD and TG again. Its diffractogram was
considered to be identical to that of Form I form, and
no significant decrease was detected in the sample
mass up to 120 °C during the TG measurement (see
Figs. S1–S2 in the Supplementary Material). It means
that the drying of the bisoprolol fumarate hydrate form
results in a solid-state transformation into the anhy-
drous Form I polymorph.
No significant decrease was observed in the sample
mass for Form I and Form II forms in the whole studied
range. It shows that these modifications are hydrate and
3000
Form I
2000
Form II
1000
2. In the second process, heat treatment was carried out
under milder conditions. Sample was heated up to
50 °C and held for 60 min at this temperature value.
The detected mass decrease was 1.9% again. The
X-ray powder diffractogram taken after the heating
process was considered to be identical to that for the
starting hydrate form of bisoprolol fumarate. TG
Hydrate
30
0
5
10
15
20
25
2θ/°
Fig. 2 X-ray powder diffraction patterns for polymorphic (Form I
and Form II) and pseudopolymorphic (hydrate) forms of bisoprolol
fumarate substance
123

Polymorphic forms of bisoprolol fumarate: preparation and characterization
Table 1 Assignment of the characteristic absorption peaks in Fourier transform infrared spectra taken from polymorphic (Form I and Form II)
and pseudopolymorphic (hydrate) forms of bisoprolol fumarate substance
Functional group/assignment
Group frequency/cm-1
Form I
Form II
Hydrate
m (O–H) stretching vibration (free)
–
–
3505
m (O–H) stretching vibration (H-bonded)
m (NH?2 ) and m (NH) stretching vibrations of associated amine
m (= CH), mas (CH3), mas (CH2), ms (CH3),
ms (CH2) stretching vibrations in chains and ring
m (C=C) aromatic skeletal vibration
3300–2200
3300–2200
3300–2200
3037, 2971, 2913, 2856 3042, 2974, 2908, 2861 2973, 2866
1610, 1512
1566
1611, 1509
1577, 1548
1462
1611, 1587
1530, 1513
1466
mas (COO-) stretching vibration
bs (CH2) scissoring vibration
1477
das (CH3) bending vibration
b (OH) deformation vibration,
ms (COO-) stretching vibration
1383, 1366, 1343, 1331 1394, 1360
1363, 1349
1232
m (C–O(H)) stretching vibration
1239, 1210
1248, 1232, 1202
mas (COC) stretching vibration (aliphatic–aromatic ether)
mas (COC) stretching vibration (aliphatic ether)
ms (COC) stretching vibration (aliphatic–aromatic ether)
cas (=CH) bending vibration of ‘‘E’’ isomer
* 1070
1043, 1029
981
1099
1121, 1090
1046
1047
978
971
c (=CH) out of-plain CH bending vibration of 1,4-disubstituted aromatic ring 855
828, 814
834, 804
Differential scanning calorimetry (DSC)
0.12
0.08
0.04
0.00
– 0.04
100.0
99.5
99.0
98.5
98.0
DSC studies were also carried out for each crystalline form
(see Fig. 5). Samples were heated up to 120 °C with a
heating rate of 10 °C min-1. Three parallel measurements
were taken.
In case of Form I and Form II samples, standard sealed
pans were used and no relevant signals were detected in the
DSC curves before the melting. Onset values of melting
points and enthalpy of fusion values are displayed in
Fig. 5.
Form II
Form I
Hydrate
Deivative curve (Hydrate)
Because of its water content, hydrate form was studied
in standard open pans. At the beginning stage of the DSC
curve, a broad endothermic peak could be observed with a
low intensity (between about 30 and 75 °C, see its enlarged
view in the inset of Fig. 5) which was followed by another,
much sharper and more intensive endothermic peak with an
onset temperature value of 76.3 ( 1.0) °C. Considering
the results of TG analysis, these two peaks probably rep-
resented the reversible and irreversible evaporation of
water, respectively: only the second process resulted in
significant change in the crystal structure (see ‘‘Thermo-
gravimetric analysis (TG)’’ section). DTG curve also
showed that the dehydration process consists of more steps,
which is in agreement to DSC results. The next endother-
mic peak represented the melting of the sample, and its
onset temperature was 101.3 ( 0.1) °C. This value shows
20
40
60
80
100
120
Temperature/°C
Fig. 4 TG curves for polymorphic (Form I and Form II) and
pseudopolymorphic (hydrate) forms of bisoprolol fumarate substance
measurement showed 1.5% of mass loss between 30
and 120 °C (see Figs. S1-S2 in the Supplementary
Material). These results indicate that the crystal
structure of the hydrate did not collapse during the
heat treatment due to the leaving of water molecules.
Probably a metastable dehydrated form was evolved,
which picked back water spontaneously from the
moisture content of the environment. So the evapora-
tion of water was found to be a reversible process
under these conditions.
123

Á. Detrich et al.
20
15
10
5
(a)
Desorption 1
Adsorption
Tm: 96.6 ( 0.1) °C
Form II
ΔrH: 123.5 ( 1.5) Jg–1
5
0
Desorption 2
Form I
Tm: 101.5 ( 0.2) °C
ΔrH: 113.1 ( 2.0) Jg–1
– 5
– 10
– 15
Hydrate
Tm: 101.3 ( 0.1) °C
ΔrH: 105.2 ( 1.0) Jg–1
20
40
60
80
100
420
0
Temperature/°C
0
20
40
60
80
400
Relative humidity/%
Fig. 5 DSC curves for polymorphic (Form I and Form II) and
pseudopolymorphic (hydrate) forms of bisoprolol fumarate substance.
(Curves were shifted in order to separate them visually.) Melting
point (Tm) and enthalpy of fusion (DrH) data are displayed for each
form. (Standard deviation values are in brackets, n = 3.) Inset shows
an enlarged view from the marked part of the curve for the hydrate
form
20
(b)
Desorption 1
Adsorption
Desorption 2
15
10
5
very good agreement to the melting point of the Form I
form, which confirms our previous assumption that the
hydrate form transforms into this crystalline modification
by its water loss at higher temperature.
Dynamic vapor sorption (DVS) measurements
Form I, Form II and hydrate forms of bisoprolol fumarate
were studied by the DVS method. The anhydrous forms of
the substance (Form I and Form II) showed very similar
behavior (see Fig. 6 and Fig. S3 in the Supplementary
Material): in the first desorption stage (40%?0% RH), no
significant mass change was observed and neither was in
the sorption stage until reaching the relative humidity value
of 80%. In this range (0–80% RH), water uptake stayed
below 0.2% for both forms, i.e., they did not show
hygroscopic property. Above 80% of relative humidity, a
significant mass gain was detected (Form I: 2.9% and
Form II: 2.4–2.6%, respectively), probably due to the for-
mation of a hydrate structure. Further increase in the rel-
ative humidity to 95% results in a huge mass gain (Form I:
17.7% and Form II: 18.0%, respectively). In this stage
presumably, the dissolution of the sample occurs, followed
by a recrystallization during the desorption process. In this
stage (95%?40% RH), the mass change value stabilized at
2.7–2.9% for both crystalline forms, which indicates that a
hydrate was formed again in the course of the recrystal-
lization process and it remained stable until the end of the
first cycle (i.e., it did not transform back to the starting or
any other anhydrous form of the substance). The second
measurement cycle clearly differed from the first one;
0
0
20
40
60
80
400
Relative humidity/%
Fig. 6 DVS sorption isotherms for bisoprolol fumarate Form I
sample (a first and b second measurement cycles)
however, it was found to be practically the same for the
two anhydrous polymorphs of the substance. In the first
desorption stage (below 20% of relative humidity), the
mass started to decrease continuously (2.0–2.1%?0.0%).
The evolved dehydrated state was not found to be
stable since the sample mass started to increase again
immediately in the sorption stage and reached the value of
2.0–2.1% at 90% RH (20% RH: 1.0%; 50% RH: 1.5% and
80% RH: 1.7–1.8%, respectively). Under extremely humid
conditions (95% RH), the dissolution of the sample hap-
pened again, attended by an extraordinary mass gain
(Form I: 15.0% and Form II: 17.1%, respectively). In the
desorption stage, the mass change value resumed promptly
below 3.0% and stabilized at 2.1–2.2%.
In case of the hydrate form, there was no significant
difference between the two measurement cycles and they
were found to be very similar to the second cycle of the
123

Polymorphic forms of bisoprolol fumarate: preparation and characterization
anhydrous forms (see Fig. S4 in the Supplementary
Material). It confirms our previous supposition that a
hydrate was formed both from Form I and Form II during
the first measurement cycle.
to the heat-of-fusion rule by Burger and Ramberger [16]. It
means that the thermodynamical stability relations reverse
at a defined temperature value.
1:1 mixture of Form I and Form II bisoprolol fumarate
polymorphs was suspended in acetone at different tem-
perature values (0, 25 and 50 °C) and stirred for 1 day. At
the end of the process, samples were filtered and studied by
XRPD. Results showed that diffractograms of the samples
stirred at 0 and 25 °C were found to be identical to that of
Form II as the sample stirred at 50 °C contained only the
Form I polymorph of the substance. This observation
indicates that the transition temperature lies between 25
and 50 °C; therefore, experiments were repeated at 35, 40
and 45 °C. After one day of stirring in acetone, samples
were filtered and studied by XRPD. Samples stirred at 35
and 40 °C were found to be identical to the Form II
polymorph as the sample stirred at 45 °C contained mainly
the Form I form with a very small amount of Form II
polymorphic contamination. It shows that at 45 °C For-
m II?Form I is the direction of the transformation; pre-
sumably, the stirring time was not enough to the complete
conversion. The difference between the stabilities (solu-
bilities) of the two polymorphic forms is probably smaller
at this temperature value, which resulted in a slower
transformation process (see Fig. S7 in the Supplementary
Material for the diffractograms).
All of the samples were studied by XRPD after finishing
the DVS measurements (see Fig. S5 in the Supplementary
Material). Diffractograms were found to be the same for all
three samples (starting materials: Form I, Form II and
hydrate forms of bisoprolol fumarate, respectively), and
they were identical to that of the hydrate form, reinforcing
the supposed polymorphic transformations based on the
sorption isotherms.
It can be stated that both anhydrous forms of bisoprolol
fumarate substance (Form I and Form II) are stable in a
broad humidity range (0–80% RH). Under extremely
humid conditions ([ & 80% RH), they transform into the
known hydrate form and at 95% RH dissolve and recrys-
tallize as the same hydrate. Hydrate was found to be stable,
too; however, it loses its water content under extremely dry
conditions (\ & 20% RH), probably forming
a
metastable dehydrate. It transformed back to the starting
hydrate form spontaneously as the relative humidity started
to increase. In this case, probably the same solid-state
transformation processes take place as it was observed by
the isothermal drying of the hydrate carried out under mild
conditions (50 °C) using the TG instrument (see ‘‘Ther-
mogravimetric analysis (TG)’’ section). During the DVS
studies only one hydrate form of the substance was iden-
tified, which is probably a monohydrate (molar ratios: 2:1:1
for bisoprolol base, fumaric acid and water, respectively).
In summary, it can be stated that the enantiotropic point
is between 40 and 45 °C: below this temperature Form II,
above this value Form I is the stable polymorph of biso-
prolol fumarate substance from the thermodynamic point
of view.
Thermodynamic stability of bisoprolol fumarate
polymorphs
Since it was proven that Form I is the stable polymorph
of the substance above 40–45 °C, DSC measurements were
taken for studying the potential Form II?Form I trans-
formation at higher temperature values. For this purpose,
Form II samples were analyzed with moderate heating rate
values (2, 1 and 0.5 °C min-1, respectively). Aside the
endothermic peak related to the melting of Form II (onset
temperature: 96.4–96.5 °C), another endothermic peak
appears in all of the DSC curves with an onset temperature
of 101.0–101.1 °C (see Fig. 7). Between these peaks, an
exothermic peak was also observed (especially in the DSC
curves recorded with lower heating rates). This endother-
mic–exothermic–endothermic peak triplet probably repre-
sents the melting of Form II form, followed by the
crystallization of Form I form from the melt and finally the
melting of the crystallized Form I form. Measured melting
points also confirmed this hypothesis (see Fig. 5). The
intensity of the exothermic and the second endothermic
peak as well increased by reducing the heating rate. The
results show that Form I polymorph of bisoprolol fumarate
can crystallize from the melt of Form II form, and the
Thermodynamic stability of bisoprolol fumarate poly-
morphs was investigated via the competitive slurry exper-
iment [35]. The 1:1 mixture of Form I and Form II
polymorphs was suspended in six different solvents (ethyl-
acetate, isopropanol, tetrahydrofuran, ethanol, diethyl-ke-
tone and acetone) and stirred for 1 day at room temperature
(25 °C). Filtered samples were studied by XRPD. At the
end of the experiment, only the Form II modification was
detectable in the samples, and Form I was disappeared in
all cases, indicating a complete Form I?Form II transition
(see Fig. S6 in the Supplementary Material). It clearly
shows that Form II is the thermodynamically stable (less
soluble) form of the substance at room temperature.
The temperature dependence of stability relations was
also studied. Previously performed DSC measurements
showed that the polymorph with a lower melting point
(Form II) has a higher enthalpy of fusion (see Fig. 5),
which indicates an enantiotropic relationship between
Form I and Form II forms of bisoprolol fumarate according
123

Á. Detrich et al.
transformation is increased by the decrease in pH. This
component can be separated by the used HPLC method.
The high recovery percentages show that the measured
solubility values do not represent the real thermodynamic
solubility: the latter value is definitely higher. The about
10% loss in recovery detected at the pH value of 1.0 (see
Table 2) can be explained by the degradation of bisoprolol
in acidic environment. pH value was checked at the end of
each measurement, and it did not change significantly
during the solubility tests.
1
0
– 1
– 2
– 3
– 4
0.5 °C min–1
1.0 °C min–1
2.0 °C min–1
For the classification of the solubility of each polymorph
dose/solubility ratio (q) and Vmax (volume in which the
highest dose strength is dissolved), values were calculated
using Eqs. 1, 2 (M: maximum dose; Smin: minimum solu-
bility over the physiological pH range; V: volume of gastric
fluid):
85
90
95
100
105
110
115
120
Temperature/°C
Fig. 7 DSC curves for bisoprolol fumarate Form II samples recorded
with different heating rate values
M
q ¼
;
ð1Þ
ð2Þ
Smin à V
effectiveness of the process can be improved by slowing
down the heating procedure.
M
Vmax
¼
:
The potential polymorphic transformations between the
presented forms are collected in Fig. 8.
Smin
The maximum dose (M) for the bisoprolol fumarate
substance is 10 mg [38]. According to the BCS, a drug
substance is considered highly soluble when the highest
dose strength is soluble in 250 mL (volume of gastric fluid)
or less of aqueous media over the pH range of 1.0–6.8 [15].
Based on these data and the measured solubility values, the
following results were calculated (see Table 5).
Solubility of bisoprolol fumarate polymorphs
The equilibrium solubility values for all the three known
crystalline forms of bisoprolol fumarate substance (Form I,
Form II and hydrate) were measured at 37 °C in three
different dissolution media with the pH values of 1.0, 4.5
and 6.8, respectively. Results are collected in Tables 2–4.
It has to be mentioned that according to our experience
under lower than neutral pH conditions, bisoprolol sub-
stance can be transformed to Bisoprolol Impurity A ((2RS)-
1-(4-hydroxymethyl-phenoxy)-3-isopropylaminopropan-2-
ol; relative retention time & 0.42). The degree of
It can be seen that the solubility properties of the three
studied bisoprolol fumarate forms do not differ signifi-
cantly in the physiological pH range, and all of them are
unambiguously highly soluble (BCS Class I or III).
Fig. 8 Potential polymorphic
transformations between the
crystalline forms of bisoprolol
fumarate and their
circumstances (dehydrate form
was not isolated: its existence is
not proven; HT: heat treatment,
RH: relative humidity)
slurry stirring (≤ 40 °C)
Form I
Form II
slurry stirring (≥ 45 °C)
HT (≈ 80 °C)
humid conditions
(RH > ≈ 80%)
humid conditions
(RH > ≈ 80%)
Hydrate
spontaneously
(under ambient
conditions)
mild HT (≈ 50 °C)
dry conditions (RH < ≈ 20%)
Dehydrate
123

Polymorphic forms of bisoprolol fumarate: preparation and characterization
Table 2 Solubility of bisoprolol fumarate polymorphs in 0.1 M HCl
solution (Mean represents the average values for the three parallel
measurements; standard deviation values are in brackets; recovery
means the percentage of the dissolved amount compared to the
weighed amount; Vmax is the volume in which the highest dose
strength (10 mg for bisoprolol fumarate) is dissolved.)
Polymorphic form
Results
Vmax mL
Amount/mg
Weighed
Recovery/%
Mean
Solubility/mg mL-1
pH (end)
1.08
Dissolved
Dissolved (mean)
9.45 ( 0.27)
Form I
Form II
Hydrate
10.167
10.840
10.341
10.857
10.779
10.474
10.487
10.360
10.587
9.24
9.75
9.35
9.92
9.88
9.54
9.39
9.25
9.52
90.4 ( 0.48)
91.4 ( 0.31)
89.6 ( 0.32)
> 0.945
\ 11
\ 11
\ 11
9.78 ( 0.21)
9.39 ( 0.13)
> 0.978
> 0.939
1.08
1.09
Table 3 Solubility of bisoprolol fumarate polymorphs in pH = 4.50
0.05 phosphate buffer solution (Mean represents the average
values for the three parallel measurements; standard deviation values
are in brackets; recovery means the percentage of the dissolved
amount compared to the weighed amount; Vmax is the volume in
which the highest dose strength (10 mg for bisoprolol fumarate) is
dissolved.)
Polymorphic form
Results
Vmax mL
Amount/mg
Weighed
Recovery/%
Mean
Solubility/mg mL-1
pH (end)
4.71
Dissolved
Dissolved (mean)
10.27 ( 0.22)
Form I
Form II
Hydrate
10.220
10.604
10.339
10.385
10.336
10.314
10.836
10.411
11.499
10.06
10.49
10.27
10.28
10.32
10.33
10.62
10.25
11.35
98.9 ( 0.46)
99.7 ( 0.57)
98.4 ( 0.32)
> 1.027
\ 10
\ 10
\ 10
10.31 ( 0.02)
10.74 ( 0.56)
> 1.031
> 1.074
4.70
4.70
Table 4 Solubility of bisoprolol fumarate polymorphs in pH = 6.80
0.05 phosphate buffer solution (Mean represents the average
values for the three parallel measurements; standard deviation values
are in brackets; recovery means the percentage of the dissolved
amount compared to the weighed amount; Vmax is the volume in
which the highest dose strength (10 mg for bisoprolol fumarate) is
dissolved.)
Polymorphic form
Results
Vmax mL
Amount/mg
Weighed
Recovery/%
Mean
Solubility/mg mL-1
pH (end)
6.81
Dissolved
Dissolved (mean)
10.17 ( 0.25)
Form I
Form II
Hydrate
9.904
10.397
10.399
10.433
10.936
10.614
10.371
10.558
10.725
9.88
10.33
10.30
10.25
10.82
10.51
10.14
10.29
10.48
99.4 ( 0.37)
98.8 ( 0.41)
97.7 ( 0.17)
> 1.017
\ 10
\ 10
\ 10
10.53 ( 0.28)
10.30 ( 0.17)
> 1.053
> 1.030
6.81
6.81
123

Á. Detrich et al.
Table 5 Dose/solubility classification of bisoprolol fumarate polymorphs (q: dose/solubility ratio; Vmax: volume in which the highest dose
strength is dissolved)
Polymorphic form
Maximum dose/mg
10
q
Vmax/mL
BCS solubility class
Dose/solubility volume
Form I
Form II
Hydrate
0.042
0.041
0.043
10.58
10.22
10.65
Highly soluble (Vmax \ 250 mL)
High solubility (q \ 1)
Competitive slurry experiments demonstrated that
Form I and Form II are in enantiotropic relationship: below
the temperature value of ca. 40–45 °C Form II, above that
Form I is the thermodynamically stable polymorph of
bisoprolol fumarate substance. DSC studies additionally
confirmed that Form I can crystallize from the melt of
Form II at high temperature.
Conclusions
The aim of this work was to study the polymorphic and
pseudopolymorphic forms of bisoprolol fumarate drug
substance. Three different crystalline forms (two poly-
morphs called Form I and Form II and a hydrate) of the
API were prepared and analyzed by various solid-state
analytical methods (XRPD, FT-IR, TG, DSC and DVS).
Thermodynamic stability and solubility properties of the
prepared forms were also investigated.
Solubility studies were also carried out investigating all
three forms at 37 °C and at three different pH values (1.0;
4.5 and 6.8, respectively). Results showed that all of the
presented polymorphs are highly soluble under all of the
studied conditions: no significant deviation was found
between the behavior of the different crystalline forms.
Experienced high solubility of the API is in agreement to
literature data; however, solubility of different polymorphs
of this substance has never been studied so far.
XRPD and FT-IR measurements showed that both
powder diffractograms and IR spectra of the three different
forms are clearly distinguishable from each other, i.e., both
techniques are suitable for the characterization of these
crystal structures.
TG and DSC analyses prove that Form I and Form II are
water and solvent free forms of bisoprolol fumarate with
melting points of 101.5 °C for Form I and 96.6 °C for
Form II, respectively. Water content of the hydrate could
be removed via isothermal heat treatment at 80 °C result-
ing in a solid-state transformation into the anhydrous
Form I form. Carrying out the heat treatment under milder
conditions (at 50 °C), water evaporation was found to be
reversible as the dried sample (possibly a metastable de-
hydrate form) took back the water from air moisture
forming a hydrate again (identical to the starting structure).
Mass loss data detected during TG measurements indicated
that the prepared pseudopolymorphic form of the API is a
monohydrate (molar ratios: 2:1:1 for bisoprolol base,
fumaric acid and water, respectively).
On the whole, two anhydrous forms and a hydrate of
bisoprolol fumarate were studied in this work. Though
significant differences were found between their physical
or physicochemical properties (melting point, thermody-
namic stability, behavior under humid conditions), due to
their uniformly high solubility it is not necessary to specify
the crystal polymorphism of the substance. Considering the
recommendations of the ICH guideline, all of the three
presented crystalline forms can be suitable for the devel-
opment of a drug product with predictable and well-defined
bioavailability and characteristics since the detected dif-
ferences do not affect the safety, performance or efficacy of
drug product.
´
Acknowledgements The authors would like to thank to Zoltan Varga
According to DVS data, Form I and Form II forms were
stable and non-hygroscopic between 0 and 80% of relative
humidity at room temperature (25 °C). Under extremely
humid conditions ([ 80% RH), both forms turned into the
known monohydrate structure. The hydrate remained
stable and lost its water content only under dry conditions
(\ 20% RH). Possibly a metastable dehydrate formed at
this point which transformed back to the hydrate immedi-
ately as relative humidity increased over 0%. At 95% of
relative humidity, all three forms dissolved and as relative
humidity decreased again below 95% always the presented
hydrate recrystallized (irrespectively of the starting crystal
structure).
} }
for his useful advices, to Gyozo Csonka-Kis, Fanni Fekete and
Gyorgyi Benene Csuta for their help in the experimental work, to
´ ´
¨
´
´
´
´
Rozalia Lıdia Vanyur and Peter Gabor Tonka-Nagy for their contri-
bution in the solubility studies and to Attila Virag for the technical
´
support.
References
1. Borka L, Haleblian JK. Crystal polymorphism of pharmaceuti-
cals. Acta Pharm Jugosl. 1990;40:71–94.
2. Lee AY, Erdemir D, Myerson MS. Crystal polymorphism in
chemical process development. Annu Rev Chem Biomol.
2011;2:259–80.
123

Polymorphic forms of bisoprolol fumarate: preparation and characterization
3. Haleblian JK, McCrone W. Pharmaceutical applications of
polymorphism. J Pharm Sci. 1969;58:911–29.
transition behaviors of gestodene polymorphs and amorphous
phase. J Therm Anal Calorim. 2017;127:1533–42.
4. Grant DJW. Theory and origin of polymorphism. Drugs Pharm
Sci. 1999;95:1–33.
21. Strickland WA. Study of water vapour sorption by pharmaceu-
tical powders. J Pharm Sci. 1962;51:310–4.
5. Fabbiani FPA, Pulham CR. High-pressure studies of pharma-
ceutical compounds and energetic materials. Chem Soc Rev.
2006;35:932–42.
22. Giron D, Goldbronn C, Mutz M, Pfeffer S, Piechon P, Schwab P.
Solid state characterizations of pharmaceutical hydrates. J Therm
Anal Calorim. 2002;68:453–65.
6. Karpinski PH. Polymorphism of active pharmaceutical ingredi-
ents. Chem Eng Technol. 2006;29:233–8.
7. Brittain HG. Polymorphism and solvatomorphism. J Pharm Sci.
2011;100:1260–79.
8. Brittain HG, Fiese EF. Effects of pharmaceutical processing on
drug polymorphs and solvates. Drugs Pharm Sci.
1999;95:331–61.
23. Brittain HG. The impact of polymorphism on drug development:
a regulatory viewpoint. Am Pharm Rev. 2000;3(67–68):70.
24. Pfeffer-Hennig S, Piechon P, Bellus M, Goldbronn C, Tedesco E.
Physico-chemical characterization of an active pharmaceutical
ingredient. J Therm Anal Calorim. 2004;77:663–79.
25. Giron D, Mutz M, Garnier S. Solid-state of pharmaceutical
compounds. J Therm Anal Calorim. 2004;77:709–47.
26. ICH Harmonized Tripartite Guideline; Specifications: test pro-
cedures and acceptance criteria for new drug substances and new
drug products: chemical substances. Q6A. 1999.
9. Chieng N, Rades T, Aaltonen J. An overview of recent studies on
the analysis of pharmaceutical polymorphs.
Biomed. 2011;55:618–44.
J Pharmaceut
10. Hilfiker R. Polymorphism: in the pharmaceutical industry.
Weinheim: Wiley-VCH Verlag GmbH & Co. KGaA; 2006.
11. Rosenstein S, Lamy PP. Polymorphism. Am J Hosp Pharm.
1969;26:598–601.
12. Brittain HG, Grant DJW. Effects of polymorphism and solid-state
solvation on solubility and dissolution rate. Drugs Pharm Sci.
1999;95:279–330.
27. Giron D. Monitoring of polymorphism—from detection to
quantification. Eng Life Sci. 2003;3:103–12.
28. Jonas R, Becker KH, Enenkel HJ, Minck K, Schliep HJ. Phe-
noxy-amino-propanole und Verfahren zu ihrer Herstellung. 1976.
DE2645710 A1.
29. European Pharmacopoeia (Ph. Eur.) 9th Edition (9.0), Mono-
graphs, Bisoprolol Fumarate; 2016.
¨
13. Amidon GL, Lennernas H, Shah VP, Crison JR. A theoretical
30. U. S. Pharmacopoeia National Formulary (USP 40 NF 35), USP
Monographs, Bisoprolol Fumarate; 2017.
basis for a biopharmaceutics drug classification: the correlation of
in vitro drug product dissolution and in vivo bioavailability.
Pharm Res. 1995;12:413–20.
31. Ramirez E, Laosa O, Guerra P, Duque B, Mosquera B, Borobia
AM, Lei SH, Carcas AJ, Frias J. Acceptability and characteristics
of 124 human bioequivalence studies with active substances
classified according to the Biopharmaceutic Classification Sys-
tem. Br J Clin Pharmacol. 2010;70:694–702.
32. Gureeva SM. Bioavailability research of solid medicinal forms by
the example of bisoprolol fumarate tablets in relation to the
biopharmaceutical classification system. News Pharm.
2014;2:10–3.
33. CharooNA, ShamsherAAA, LianLY, Abrahamsson B, Cristofoletti
R, Groot DW, Kopp S, Langguth P, Polli J, Shah VP, Dressman J.
Biowaiver monograph for immediate-release solid oral dosage
forms: bisoprolol fumarate. J Pharm Sci. 2014;103:378–91.
34. Greenspan L. Humidity fixed points of binary saturated aqueous
solutions. J Res NBS A Phys Chem. 1977;81A:89–96.
35. Mangin D, Puel F, Veesler S. Polymorphism in processes of
crystallization in solution: a practical review. Org Process Res
Dev. 2009;13:1241–53.
14. Waiver of In Vivo Bioavailability and Bioequivalence Studies for
Immediate-Release Solid Oral Dosage Forms Based on a Bio-
pharmaceutics Classification System (Guidance for Industry).
U. S. Department of Health and Human Services, Food and Drug
Administration, Center for Drug Evaluation and Research
(CDER). 2017. https://www.fda.gov/downloads/Drugs/Guidance
ComplianceRegulatoryInformation/Guidances/UCM070246.pdf.
Accessed 17 Jan 2018.
15. Yu LX, Amidon GL, Polli JE, Zhao H, Mehta MU, Conner DP,
Shah VP, Lesko LJ, Chen ML, Lee VHL, Hussain AS. Bio-
pharmaceutics classification system: the scientific basis for bio-
waiver extensions. Pharm Res. 2002;19:921–5.
16. Burger A, Ramberger R. On the polymorphism of pharmaceuti-
cals and other molecular crystals. I. Theory of thermodynamic
rules. Microchim Acta. 1979;2:259–71.
17. Burger A, Ramberger R. On the polymorphism of pharmaceuti-
cals and other molecular crystals. II. Applicability of thermody-
namic rules. Microchim Acta. 1979;2:273–316.
´
´
36. Baka E, Comer JEA, Takacs-Novak K. Study of equilibrium
solubility measurement by saturation shake-flask method using
hydrochlorothiazide as model compound. J Pharm Biomed.
2008;46:335–41.
18. Burger A. Thermodynamic and other aspects of the polymor-
phism of drugs. Pharm Int. 1982;3:158–63.
´
37. Dinya Z. Infrared spectroscopy (in Hungarian). Tankonyvkiado:
Kossuth Lajos University (Debrecen), Faculty of Natural Sci-
ences; 1981.
38. https://www.drugs.com/dosage/bisoprolol.html. Accessed 17 Jan
2018.
¨
19. Djellouli F, Dahmani A, Hassani A. Characterization of the
polymorph changes in Trimethoprim. J Therm Anal Calorim.
2017;130:1585–91.
20. Wang LY, Zhu L, Sha ZL, Li XC, Wang YF, Yang LB, Zhao XY.
Characterization, thermal stability, and solid-state phase
123