European Journal of Pharmaceutical Sciences 65 (2014) 5664

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European Journal of Pharmaceutical Sciences

journal homepage: www.elsevier.com/locate/ejps

Cocrystal screening of hydroxybenzamides with benzoic acid

derivatives: A comparative study of thermal and solution-based
methods
Alex N. Manin a, Alexander P. Voronin a, Ksenia V. Drozd b, Nikolay G. Manin a, Annette Bauer-Brandl c,
German L. Perlovich a,
a
G.A. Krestov Institute of Solution Chemistry of the Russian academy of Sciences, 1, Akademicheskaya, 153045 Ivanovo, Russian Federation
b
Ivanovo State University, 39, Ermaka, 153025 Ivanovo, Russian Federation
c
Department of Physics, Chemistry and Pharmacy, University of Southern Denmark, Campusvej 55, 5230 Odense M, Denmark

a r t i c l e i n f o a b s t r a c t

Article history: The main problem occurring at the early stages of cocrystal search is the choice of an effective screening
Received 19 July 2014 technique. Among the most popular techniques of obtaining cocrystals are crystallization from solution,
Received in revised form 29 August 2014 crystallization from melt and solvent-drop grinding. This paper represents a comparative analysis of the
Accepted 3 September 2014
following screening techniques: DSC cocrystal screening method, thermal microscopy and saturation
Available online 16 September 2014
temperature method. The efciency of different techniques of cocrystal screening was checked in 18
systems. Benzamide and benzoic acid derivatives were chosen as model systems due to their ability to
Keywords:
form acid-amide supramolecular heterosynthon. The screening has conrmed the formation of 6 new
Screening
Solubility
cocrystals. The screening by the saturation temperature method has the highest screen-out rate but
Melting process the smallest range of application. DSC screening has a satisfactory accuracy and allows screening
Cocrystal over a short time. Thermal microscopy is most efcient as an additional technique used to interpret
ambiguous DSC screening results. The study also included an analysis of the inuence of solvent type
and component solubility on cocrystal formation.
2014 Elsevier B.V. All rights reserved.

1. Introduction cocrystals) allows us to change physicochemical and

In the last few decades, pharmaceutical industry has achieved a
great progress in its search for new drug compounds by applying
combinatorial approaches and high throughput in vivo screening.
However, compounds obtained by such methods typically have a
number of defects, the main one being their low solubility in
aqueous media and, consequently, their low bioavailability. 70%
of the compounds under development and 40% of the drugs on
the market have poor water solubility (Thayer, 2010). Therefore,
creating soluble drug compounds by using innovative techniques
is now becoming one of the most urgent tasks.
Cocrystallization is a promising approach increasing the
bioavailability of active pharmaceutical ingredients (API)
(Schultheiss and Newman, 2009), their thermodynamic stabil-
ity (Vishweshwar et al., 2006) and a wide range of mechanical
properties (Sun and Hou, 2008; Karki et al., 2009). Besides,
using coformers of different nature (as a second component of
Corresponding author. Tel.: +7 4932 533784; fax: +7 4932 336237.
E-mail address: glp@isc-ras.ru (G.L. Perlovich).
pharmacokinetic properties of the system, thus making it
possible to ne-tune products under development to market
requirements (Shan and Zaworotko, 2008). Cocrystals are also
interesting as potential intellectual property items which can
bring back into the market generic drugs with improved
characteristics as a unique brand (Trask, 2007).
The literature describes a lot of approaches to obtaining
cocrystals (Karki et al., 2007; Rodrguez-Hornedo et al., 2006;
Padrela et al., 2009; Daurio et al., 2011; Eddleston et al., 2013;
Alhalaweh and Velaga, 2010; Morrison et al., 2013; Oswald and
Pulham, 2008), as well as analytical methods suitable for their
identication (Trask et al., 2005; Alles et al., 2008; Elbagerma
et al., 2010; Maruyoshi et al., 2012). Unfortunately, there are still
no universal rules for selection of more suitable algorithms for
cocrystal screening of preassigned system. The choice of a screen-
ing method depends on the problem to be solved, the nature of the
object of research (such as the difference in solubility of API and
coformers in the used solvents, thermal stability or aptness to form
stable solvates) and availability of sufcient amount of the
substance. The strengths and weaknesses of many screening

http://dx.doi.org/10.1016/j.ejps.2014.09.003
0928-0987/ 2014 Elsevier B.V. All rights reserved.
 A.N. Manin et al. / European Journal of Pharmaceutical Sciences 65 (2014) 5664
methods have not been objectively analyzed yet, though they
determine the methods real application range. As new approaches
appear, it becomes more and more difcult to choose among them
the most suitable one for certain research objects. Therefore, it is
necessary to develop a number of criteria of comparing the ef-
ciency of different cocrystal screening techniques.
Using powder X-ray diffraction analysis (PXRD) can be suitable
for quick identication of the substance and its cell parameters
(Trask et al., 2005). However, this technique is not suitable for
full-scale high throughput screening if it is used as the only
identication technique (Alles et al., 2008) because in this case
samples should be additionally treated by neat grinding, solvent-
drop grinding or other cocrystal preparation techniques. Therefore,
our study was aimed at conducting comparative analysis of the
practical efciency of a number of alternative methods currently
used in pharmaceutical cocrystal screening.
The screening procedure of selecting pharmaceutical cocrystals
with preset properties includes two consecutive stages: (a) cocrys-
tal formation and (b) identication of its properties (solubility,
thermodynamic stability, dissolution kinetics, etc.). In turn, the
methods of obtaining cocrystals can be divided into kinetic and
thermodynamic types (Newman, 2013).
Thermodynamic methods are used to obtain a stable crystal form
under the given conditions (usually this process goes under condi-
tions close to equilibrium and requires a lot of time) (Newman,
2013). Thermodynamic methods include crystallization through
slow evaporation of solvent, crystallization from melt and solu-
tion-mediated phase transformation.
Kinetic methods are most suitable for searching metastable
crystal forms with higher Gibbs energy values compared to stable
crystals. Experiment conditions for these methods are non-equilib-
rium. In some cases additional energy is supplied to overcome
energy barriers and the process usually takes minutes or even
seconds. Kinetic methods include grinding, slurry sonication and
fast solvent evaporation (such as spray-drying, crystallization from
supercritical states). Such division was used by Anderton (2007) to
classify the methods of obtaining polymorph modications.
There is a number of screening techniques that combine cocrys-
tal formation and its analysis. They include DSC screening (Lu et al.,
2008), thermal microscopy (TM) (Berry et al., 2008) and saturation
temperature method (STM) (ter Horst et al., 2009). When these
methods are used, a cocrystal is formed from a physical mixture
right during the experiment and has strictly determined character-
istics (solubility, fusion temperature and enthalpy, crystal habit,
etc.). As such methods can increase screening applicability, in this
work we analyze and compare their efciency in a number of
model binary systems.
Benzoic acid and benzamide derivatives capable of forming a
stable acid-amide supramolecular heterosynthon (Shan and
Zaworotko, 2008) (Fig. 1) were chosen as the research objects. 2-
hydroxybenzamide (2-OHBZA, salicylamide) and its meta- and
para-isomers (3-/4-OHBZA) were used as model compounds with
an amide group, with benzoic (BA), salicylic (SA), acetylsalicylic
(ASA), 2-,3- and 4-acetamidobenzoic ((2-/3-/4-AcAmBA) acids as
coformers (Table 1).
Salicylic, acetylsalicylic acids and 2-hydroxybenzamide are
nonsteroidal anti-inammatory drug compounds of the salicyl
series, the biological action of which is based on selective inhibi-
O H O
R1 R2
N H O ter. Then, the sample mixtures corresponding to 1 ml of saturated
H solution of both components at t0 were prepared and the said
Fig. 1. Acidamide heterosynthon.
57
tion of cyclooxygenase enzyme (COX-1 and COX-2) catalyzing
prostaglandin synthesis (Rainsford, 2004). The literature shows
that benzoic acid and its derivatives form cocrystals with many
drug compounds and widely used coformers (Seaton and Parkin,
2011), such as carbamazepine (Childs et al., 2008), uconazole
(Kastelic et al., 2011) and anti-HIV compound didanosine (Alatas
et al., 2013). Acetylsalicylic acid is proved to form a soluble
drug-drug cocrystal with meloxicam (Cheney et al., 2011). Salicyl-
amide isomers are also used as model coformers in pharmaceutical
cocrystal screening (Tothadi and Desiraju, 2012).
2. Materials and methods
2.1. Materials
2- and 3-hydroxybenzamide (assay 98%) were purchased from
Fluka. 4-hydroxybenzamide, 3- and 4-AcAmBA (98%), BA, SA and
2-AcAmBA (P99%) were purchased from SigmaAldrich. Acetylsal-
icylic acid with minimal purity of 98% was purchased from Norsk
Medisinal Depot. Ethanol, methanol and acetone (assay 99 + %)
by CHEMMED company were used as solvents in STM screening
experiments.
All substances were used as received without additional
purication. The purity of substances was controlled by DSC.
2.2. Methods
2.2.1. Differential scanning calorimetry
DSC screening was carried out using the DSC 204 F1 Phoenix
differential scanning heat ux calorimeter (NETZSCH, Germany)
with a high sensitivity l-sensor (sensitivity 0.0025 lW (65 lV/
mW), time constant 2.3 s). The sample was heated from 25 to up
to 270 C depending on melting point of less fusible component at
the rate of 10 K  min 1 in an argon atmosphere and cooled with
gaseous nitrogen. Temperature calibration of the DSC was
performed against six high-purity substances, cyclohexane
(99.96%), mercury (99.99 + %), biphenyl (99.5%), indium
(99.999%), tin (99.999%), and bismuth (99.9995%). Through calibra-
tion experiments the temperature error was established as 0.5 K,
error in phase transition enthalpy 1%. The sample quantity in
all experiments was between 1.80 and 2.50 mg, while the accuracy
of weighting procedure was 0.01 mg.
2.2.2. Solvent-drop grinding
The grinding procedures were performed as follows: a stoichi-
ometric mixture of components was placed into the agate milling
jar of Pulverisette 7 planetary micromill and a corresponding quan-
tity (approx. 1 ll per 1 mg of mixture) of ethanol was added. Then
the mixture was ground for 30 min at 600 rpm with 10 5-mm
agate balls and the jar was left for 510 min at a room temperature
to let the solvent evaporate. The purity of produced cocrystal was
controlled by observing the endotherms on the DSC curve.
2.2.3. Saturation temperature method
STM screening was carried out according to the method
described in the article by ter Horst (ter Horst et al. (2009)) with
ethanol, methanol and acetone used as solvents. First, saturated
solutions of pure components were prepared by isothermal satura-
tion method by stirring them in an air thermostat for 2024 h at a
starting temperature t0 of 20 C and the equilibrium concentrations
were measured using the UVVis Varian Cary 50 spectrophotome-
volume of solvent was added to each of them. The temperature,
at which the mixtures have completely dissolved, was determined
58 A.N. Manin et al. / European Journal of Pharmaceutical Sciences 65 (2014) 5664

Table 1
Structural formulas and basic properties of the selected compounds.

Compound 2-OHBZA 3-OHBZA 4-OHBZA BA SA

Structural formula H H H H H
O N O N O N O O O O
H H H H H
O O

O
H O
H
M, g 1 137.14 137.14 137.14 122.12 138.12
Tm (DSC)1, C 140.8 167.6 159.9 123.1 158.9
SEtOH (20 C), mol l 1 0.51 0.01 0.41 0.02 1.48 0.02 2.88 0.06 2.13 0.02
SMeOH (20 C), mol l 1 0.739 0.007 0.90 0.02 1.94 0.02 3.89 0.03 2.36 0.02
SAcetone (20 C), mol l 1 1.48 0.02 0.29 0.02 0.124 0.006 1.98 0.03 2.08 0.04
Compound ASA 2-AcAmBA 3-AcAmBA 4-AcAmBA
Structural formula H H H H
O O O O O O O O
H
O CH3 H3C N

O O H
N

O CH3
N O
H
CH 3
M, g 1 180.10 179.18 179.18 179.18
Tm (DSC)a, C 142.0 212.4 237.2 262.3
SEtOH (20 C), mol
l 1 1.03 0.02 0.27 0.02 0.061 0.003 0.171 0.005
SMeOH (20 C), mol l 1 1.11 0.01 0.41 0.01 0.076 0.001 0.188 0.003
SAcetone (20 C), mol l 1 1.009 0.004 0.20 0.01 0.022 0.002 0.051 0.001
a
Melting points were determined experimentally as onset fusion temperatures of pure compounds at standard error of 0.2 C.

by visually observing the samples after every 45 h of stirring at
constant temperature and raising the temperature if solid particles
remained. In another variant of method, stirring was performed in
an ultrasonic bath for 30 min at 25 C.
2.2.4. Thermal microscopy
Thermal microscopy was performed using the Altami Polar 312
optical microscope equipped with MICROSTAT-N heating stage,
USB 3150R6 1/2CMOS USB camera, 4/60 objective and Altami
Studio 2.0 software kit. A powder sample of homogenized physical
mixture or cocrystal obtained by cogrinding was placed between
two glass plates of heating stage and heated at 6 C/min until
complete melting. Phase transitions were observed through the
microscope in isothermal mode. Upon melting, the sample was left
cooling and the order of crystal formation from melt was identi-
ed, as well as their shape. In case of obtaining a new phase, its
melting point and thermal stability was determined by heating
the mixture once more. Prior to the experiment, a heating/cooling
cycle was performed for individual compounds to determine the
morphology of crystals falling from melt. Melting points of pure
components are equal to the reference data with the accuracy of
1 C.
2.3. Physical characterization of cocrystals
2.3.1. X-ray powder diffraction (PXRD)
Formation of cocrystal was ensured using PXRD patterns. Pow-
der X-ray diffraction experiment was carried out on the Bruker D8
Advance X-ray diffractometer using Mo Ka radiation. The voltage
and current applied were 40 kV and 40 mA, respectively. Data were
collected in the range of 2h = 530 with a step size 0.03, scan
time for one step 3s.
3. Results and discussion
3.1. Results
3.1.1. PXRD phase analysis of cocrystals obtained by solvent-drop
grinding
18 binary systems with benzamide and benzoic acid derivatives
of different stoichiometry were checked by cocrystal screening
(Table 2); 6 new cocrystals were obtained.
The binary mixtures of the components under consideration
with the molar ratio (1:1) were ground with ethanol, dried and
studied by the X-ray powder diffraction method. Analysis of
diffraction patterns of the ground samples has shown a change
in the crystal structure compared to pure substances in 6 systems:
[2-OHBZA + BA], [2-OHBZA + SA], [2-OHBZA + 4-AcAmBA], [3-
OHBZA + 2-AcAmBA], [4-OHBZA + SA] and [4-OHBZA + 2-AcAmBA]
(Table 2). The gure shows the sample X-ray diffraction prole of
the [2-OHBZA + BA] system and individual components (Fig. 2).
New peaks caused by grinding are marked by asterisks.
It should be noted that the least active coformers among the
objects are meta-isomers (only one positive result among six
coformers with meta-hydroxybenzamide), while the most suitable
coformers for cocrystallization are compounds with substitutes in
ortho-position (ve out of six cocrystals, one of the cocrystals is
formed by two acid and amide ortho-isomers). The screening
results obtained by solvent-drop grinding/PXRD will be used fur-
ther in this paper as a benchmark to evaluate the efciency of
screening techniques on these objects.
3.1.2. DSC screening of physical mixtures
As all the compounds under study (API and coformers) melt
without decomposing, DSC screening can be used (Lu et al.,
2008). To apply this method, two individual components were
 A.N. Manin et al. / European Journal of Pharmaceutical Sciences 65 (2014) 5664 59

Table 2
DSC screening results for the API and coformers physical mixture with stoichiometry (1:1).

API Coformers DSC screening results (C) Exothermic peak (yes/no) (C) DSC after grinding results Cocrystal reportedb (yes/no)
First peak Second peak
2-OHBZA BA 112.1 Yes (100.9) Yes Yes
SA 112.3 121.6 No Yes Yes
ASA 106.9 No No No
2-AcAmBA 132.8 No No No
3-AcAmBA 134.1 No No No
4-AcAmBA 137.6 181.8 Yes (146.1) Yes Yes
3-OHBZA BA 121.4 No No No
SA 133.2 No No No
ASA 124.3 No No No
2-AcAmBAa 162.2 192.3 No No Yes
3-AcAmBA 161.7 No No No
4-AcAmBA 162.4 No No No
4-OHBZA BA 113.7 No No No
SA 152.5 165.3 Yes (157.6) Yes Yes
ASA 116.8 No No No
2-AcAmBAa 151.8 171.8 No No Yes
3-AcAmBA 156.2 No No No
4-AcAmBA 155.5 No No No
a
Stoichiometry (2:1).
b
Determined by powder X-ray diffraction experiments after solvent-drop grinding.

by two selection techniques described above. And this is mainly

Fig. 2. PXRD patterns for: 2-OHBZA (1), BA (2), [2-OHBZA + BA] (1:1) after solvent-
drop grinding (3).
mixed in stoichiometric proportions and put in crucibles. There
were distinctive melting peaks on the DSC curve, which were later
analyzed to identify the cocrystal formation using the rules sug-
gested by Lu et al. (2008) and Yamashita et al. (2013). According
to these rules, components are capable of forming cocrystals if
the following conditions are fullled: (a) the physical mixture
melting produces two peaks corresponding to eutectic mixture
and cocrystal melting (with their temperatures being different
from the melting temperatures of individual components) (Lu
et al., 2008); (b) the eutectic melting (the rst peak) is followed
by a small exo-effect (Yamashita et al., 2013).
To test the chosen screening technique and interpret its results,
we analyzed the physical mixtures of components, cocrystal for-
mation in which had been earlier described in literature (reference
systems). [Benzamide + benzoic acid] (Brittain, 2009) and [indo-
methacin + nicotinamide] (Mirza et al., 2008) were selected as such
systems. When these mixtures were melted, neither two peaks nor
exo-effect typical of cocrystallization were observed. Evidently, in
these cases the temperatures of physical mixture melting were
close to cocrystal melting temperatures (Fig. 3a and b), which led
to thermal effects overlap. Thus, DSC experiments cannot be used
to unambiguously determine the results of cocrystal formation
caused by the thermal effects overlap.
DSC screening was used to check 18 systems, three of which
showed a positive result. It should be noted that only one melting
peak was observed in case of the (2-OHBZA + BA) object (as in case
of (benzamide + benzoic acid) system (Brittain, 2009)); however,
an exo-effect indicating cocrystal formation was clearly seen
before melting (Fig. 4). The complexities of peak interpretation
for the physical mixtures (3-OHBZA + 2-AcAmBA) and (4-OHBZA
+ 2-AcAmBA) are discussed below.
The main DSC screening results are represented in Table 3.
Only one distinctive peak was observed in 12 out of 18 systems.
Cocrystal formation in these systems is unlikely but not excluded
due to overlapping thermal effects of melting of the eutectic
mixture and cocrystal. Such systems should be studied in detail
by other techniques. It is important to mention a regularity
observed while screening physical mixtures in which the
components melting temperatures differ from each other by more
than 50 C. If the melting occurred in one step with the mixture
melting temperature slightly lower than that of the fusible
component, no cocrystal was formed.
3.1.3. DSC screening of the mixture obtained by solvent-drop grinding
The next screening stage was DSC analysis of the ground mix-
ture. DSC analysis of most samples after solvent-drop grinding
has proved that no cocrystal was formed. Only in case of three sys-
tems ((2-OHBZA + SA) (1:1); (2-OHBZA + 4-AcAmBA) (1:1) and (4-
OHBZA + SA) (1:1)) the DSC analysis of the ground up mixture
showed the disappearance of the rst peak characterizing the
eutectic fusion. It means that these three systems form cocrystals
and, as a result of mechanochemical activation, the physical
mixture is completely transformed into a cocrystal (Fig. 5). After
the grinding of the (2-OHBZA + BA) physical mixture the exo-effect
disappeared from the DSC curve (Fig. 4), which can be caused by
cocrystal formation.
The X-ray diffraction analysis of the ground up systems
(3-OHBZA + 2-AcAmBA) and (4-OHBZA + 2-AcAmBA) has proved
cocrystal formation, while DSC screening of the systems in
stoichiometry (1:1) has shown two peaks during the mixture melt-
ing. However, since these mixtures were melted, the same two
peaks were observed on the DSC curve after solvent-drop grinding
60 A.N. Manin et al. / European Journal of Pharmaceutical Sciences 65 (2014) 5664

Fig. 3. DSC curves of cocrystal-forming physical mixtures: (benzamide + benzoic acid) (Brittain, 2009) (a) (benzamide (1), benzoic acid (2), a (1:1) physical mixture of
benzamide and benzoic acid (3)), (indomethacin + nicotinamide) (Mirza et al., 2008) (b) (indomethacin (1), nicotinamide (2), a (1:1) physical mixture of indomethacin and
nicotinamide (3)).

Table 3
Overall screening results obtained by solvent-drop grinding/powder X-ray diffraction
(SDG/PXRD), DSC screening, thermal microscopy (TM) and saturation temperature
method (STM).

Amide Acid SDG/PXRD DSC TM STM

2-OHBZA BA Yes Yes Yes No
SA Yes Yes Yes No
ASA No ?a No No
2-AcAmBA No ?a No No
3-AcAmBA No ?a No No
4-AcAmBA Yes Yes ?d Yes
3-OHBZA BA No ?b No No
SA No ?a No No
ASA No ?b No No
2-AcAmBA Yes ?c Yes Yes
3-AcAmBA No No No No
4-AcAmBA No No No No
Fig. 4. DSC thermograms of 2-OHBZA (1), BA (2), their (1:1) mixture (3) and 4-OHBZA BA No ?a No No
cocrystal (4) of the same ratio. Note the exothermal peak at a heating curve of the SA Yes Yes Yes Yes
mixture. ASA No ?a No No
2-AcAmBA Yes ?c Yes Yes
3-AcAmBA No No No No
as in the screening process. Therefore, the two peaks occurring in 4-AcAmBA No No No No
the process of DSC screening are interpreted in a different way. a
A peak overlap may occur, as Tm of the components differ by less than 50 C.
The rst peak on the curve corresponds to the melting of the b
The second peak appears as a shoulder.
cocrystal formed, while the second peak corresponds to the c
Positive results were obtained only for (2:1) molar ratio.
melting of excessive amount of the component with higher Tm. d
Cocrystal decomposes upon melting, resulting in precipitation of high-melting
The melting temperatures of the components of these two component from melt at the cocrystal melting point.
mixtures are close to each other, therefore, as it was shown above,
most probably there was a merge of temperatures of eutectic
fusion and cocrystal fusion in both cases. Repeated grinding of of a eutectic mixture. A ground up eutectic mixture cannot be
these mixtures with the molar ratio (2:1) with the hydroxyben- absolutely homogeneous, therefore, its different segments pass to
zamide excess has conrmed that stoichiometric proportion of a liquid state at different temperatures unlike cocrystals and pure
the components was not (1:1). (Fig. 6). compounds that melt in a uniform manner at single melting point.
Analysis of the homogeneous mixture with the compound ratio
3.1.4. Thermal microscopy of physical mixtures (1:1) revealed the formation of 5 cocrystals. In the (2-OHBZA + BA),
As DSC thermograms cannot always show whether or not a (2-OHBZA + SA) and (4-OHBZA + SA) systems the rst to melt was
cocrystal was formed, we supplemented DSC screening with a the eutectic mixture, which then resulted in the formation of more
thermal microscopy analysis (Berry et al., 2008). Thermal micros- refractory materials melting at cocrystal Tm. In (3-OHBZA + 2-AcA-
copy analysis consists in observing phase transitions occurring in mBA) and (4-OHBZA + 2-AcAmBA) mixtures, the sample melted
the system when it is heated. The following criteria indicate partially at eutectic temperature with the subsequent melting of
cocrystal formation: (a) uniform melting of the sample at a xed the second component within a wide temperature range that can
temperature; (b) cocrystallization of components from the melt; be explained by the difference of the composition of the mixture
(c) a different morphology of the obtained crystals compared to being analyzed from the cocrystal stoichiometry. Crystallization
that of the original compounds. The advantage of thermal micros- under sample cooling happened at the same time for all the
copy over DSC screening is the fact that this technique allows dis- systems in the full volume of the melt, which indicated the mixture
tinguishing the melting of a chemical compound from the melting homogeneity (Fig. 7). Repeated heating of the obtained crystals has
 A.N. Manin et al. / European Journal of Pharmaceutical Sciences 65 (2014) 5664 61

Fig. 5. DSC thermograms: (a) 2-OHBZA (1), SA (2), (2-OHBZA + SA) (1:1) physical mixture (3), [2-OHBZA + SA] (1:1) cocrystal (4); (b) 2-OHBZA (1), 4-AcAmBA (2), (2-
OHBZA + 4-AcAmBA) (1:1) physical mixture (3), [2-OHBZA + 4-AcAmBA] (1:1) cocrystal (4); (c) 4-OHBZA (1), SA (2), (4-OHBZA + SA) (1:1) physical mixture (3), [4-
OHBZA + SA] (1:1) cocrystal (4).

Fig. 6. DSC curves of: (a) 3-OHBZA (1), 2-AcAmBA (2), (3-OHBZA + 2-AcAmBA) (1:1) physical mixture (3), (3-OHBZA + 2-AcAmBA) (1:1) mixture after solvent-drop grinding
(4), (3-OHBZA + 2-AcAmBA) (2:1) mixture after solvent-drop grinding (5); (b) 4-OHBZA (1), 2-AcAmBA (2), (4-OHBZA + 2-AcAmBA) (1:1) physical mixture (3), (4-OHBZA + 2-
AcAmBA) (1:1) mixture after solvent-drop grinding (4), (4-OHBZA + 2-AcAmBA) (2:1) mixture after solvent-drop grinding (5).

Fig. 7. Crystallization of a [4-OHBZA + SA] (1:1) cocrystal from a gently cooled melt of eutectic mixture.

shown that they melt at the temperature different from that
of the mixture components but corresponding to the DSC peak
temperature of the ground mixture, i.e. make up a cocrystal.
In case of the physical mixtures (4-OHBZA + SA) (1:1) and
(3-OHBZA + 2-AcAmBA) (1:1), the morphology of the formed
crystals did not correspond to that of the crystals obtained in the
absence of a second component (Fig. 8).
In binary systems where the difference in Tm was over 60 C,
the components crystallized from melt separately. This conclusion
is conrmed by the form of the obtained crystals and their melting
temperatures under repeated heating. This indicates that it is
impossible to obtain a stable cocrystal from melt for these mix-
tures. However, the above limitation could be relied upon only
after a thorough study on a larger set of compounds.
3.1.5. Saturation temperature screening
Since the compounds under study are rather well soluble in
polar organic solvents, the saturation temperature method can be
used for screening (ter Horst et al., 2009). This screening technique
has a lot in common with the widely-spread method of obtaining
cocrystals by solution crystallization (Rodrguez-Hornedo et al.,
2006) as both of them are based on the same process of solution-
mediated phase transformation and are aimed at obtaining ther-
modynamically stable cocrystals from the supersaturated solution
of their components.
If a cocrystal is thermodynamically stable under given condi-
tions (i.e. is formed spontaneously from a solution containing a
mixture of components), its solubility is lower than that of the
mixture of a respective composition. If at the given temperature
t0 the components concentrations correspond to a saturated solu-
tion, cocrystal formation results in its precipitation, with the pre-
cipitate dissolution requiring heating to a higher temperature.
Practically, if we know the solubility of original compounds in
solution at t0, we can make a sample corresponding to a certain
volume of saturated solution of both components at this tempera-
ture and, based on the change of the saturation temperature DT in
62 A.N. Manin et al. / European Journal of Pharmaceutical Sciences 65 (2014) 5664

Fig. 8. (a) Morphology of crystals formed from a melt of (3-OHBZA + 2-AcAmBA) (2:1) physical mixture: 1 cocrystal, 2 crystals of 3-OHBZA, 3 crystals of 2-AcAmBA. The
shape of cocrystal clearly differs from crystals formed from melt of pure 3-OHBZA (b) and 2-AcAmBA (c).

Fig. 9. Saturation temperature increase (DT (STM), grey bars) and solubility ratio of components at t0 (a, black squares) for mixtures that form a precipitate in the STM
screening. It can be seen that for 3 of 4 systems low a values correspond to higher DT, and vice versa. At 42 C the screening experiment was stopped.

the respective solvent volume, determine whether a cocrystal is
formed or not (ter Horst et al., 2009). The precipitate obtained is
a pure cocrystal that can later be analyzed by other methods.
To evaluate the inuence of the solvent on the experiment
results by the saturation temperature method (STM), the screening
was conducted in three different solvents: ethanol, methanol and
acetone. Before conducting the analysis we measured the solubility
of all the compounds under study at the initial temperature
t0 = 20 C (Table 1).
The analysis showed that in 14 systems out of 18 there was no
precipitation while in (2-OHBZA + 4-AcAmBA), (3-OHBZA + 2-AcA-
mBA), (4-OHBZA + SA) and (4-OHBZA + 2-AcAmBA) solutions, pre-
cipitation in the form of disperse particles was observed. The
results showed that the maximal saturation temperature rise
was: in the (2-OHBZA + 4-AcAmBA) system 21 C in ethanol; in
the (3-OHBZA + 2-AcAmBA) system over 22 C in ethanol, meth-
anol and acetone; in the (4-OHBZA + SA) system over 22 C in
ethanol and methanol; in the (4-OHBZA + 2-AcAmBA) 14 C in
methanol. Precipitate analysis for all the samples has conrmed a
complete transformation of the components into a cocrystal.
Experimental data indicate that a solvent inuence on cocrystal
precipitation from a saturated solution of components is mainly
determined by the solubility ratio of the pure components in this
solvent. To determine this regularity we introduced parameter a
showing the correlation of equilibrium solubilities of the well-sol-
uble and the poorly-soluble components at the temperature
t0 = 20 C. The dependence of DT on a in different solvents in the
systems, producing positive results when the saturation tempera-
ture method is used, is represented in Fig. 9.
As Fig. 9 shows, low a values, as a rule, corresponded to nonzero
DT and, on the contrary, there was no precipitation in the systems
where components solubilities differ by a factor of ten and more. In
the (2-OHBZA + 4-AcAmBA) and (4-OHBZA + SA) systems an obvi-
ous dependence of a in different solvents on DT value decrease
was observed though in the (4-OHBZA + 2-AcAmBA) mixture there
was an opposite dependence.
Despite the fact that the method accuracy in relation to nega-
tive results was high, only four out of six possible cocrystals pro-
duced positive results when it was used. For the (2-OHBZA + BA)
and (2-OHBZA + SA) systems, in which the difference in compo-
nents solubility did not exceed 6 times, there was no precipitation
in any of the selected solvents. More denite applicability limits for
this method are yet to be determined on a larger number of objects
of different nature.
Sonication is often used to speed up phase transformation
processes including those of cocrystal formation (Alatas et al.,
2013; Morrison et al., 2013). Therefore, to reduce the screening
time, an experiment was conducted by the saturation temperature
method in an ultrasound bath with ethanol as a solvent. It was
determined that for all the four samples producing a positive result
(forming cocrystals) the precipitation took 30 min (according to
the bottom phase DSC results).
 A.N. Manin et al. / European Journal of Pharmaceutical Sciences 65 (2014) 5664
3.2. Discussion
3.2.1. Differential scanning calorimetry
DSC screening technique allows eliminating errors caused by
the defects of cocrystal production methods as cocrystal formation
occurs during the experiment and is detected right in the melting
process. Simplicity, speed and economic efciency of the DSC
method make it most suitable to be the rst stage of multi-stage
screening aimed at screening out the systems where components
do not interact with each other. At the same time, this method is
inapplicable to volatile and thermally unstable components.
4 systems out of 18, i.e. 22% of the total number, had a negative
result after DSC screening. All three systems with two melting
points form a cocrystal (conrmed by PXRD). A positive result
was also obtained in the (2-OHBZA + BA) system with one peak
as in [benzamide + benzoic acid] (1:1) (Brittain, 2009), [indometh-
acin + nicotinamide] (Mirza et al., 2008). For a large number of sys-
tems (55%) it was impossible to identify the presence or absence of
a cocrystal based on DSC experiment solely due to the difculty of
peak interpretation. It was shown that this uncertainty could be
partially eliminated by grinding the mixture prior to DSC analysis.
Based on the conducted experiments it was found that when
the melting temperatures of pure components differ insignicantly
(within 50 C), the cocrystal peak can partially or completely over-
lap with the melting peak of the eutectic mixture. In such cases
presence of only one peak on the melting curve does not prove that
no cocrystal is formed in the system. It is a clear illustration of why
there will not be one universal method for cocrystal screening. Dif-
ferent techniques will be more or less useful depending on the nat-
ure of the compounds, and nding the reliable conditions is one of
the goals of this paper.
If the melting temperatures difference is over 50 C, one peak on
the physical mixture DSC curve below the melting temperature of
the more volatile component denitely shows that no cocrystal
was formed in the system. This regularity allows screening out a
number of systems at the DSC screening stage without doing addi-
tional research.
3.2.2. Thermal microscopy
The conducted experiments have shown a comparatively high
efciency of thermal microscopy both in visualizing DSC results
and as an independent technique of cocrystal screening. Besides
screening, thermal microscopy also allows determining of thermal
stability of the cocrystal being formed and the possibility of obtain-
ing it from melt.
The experimental study has proved that this method is not
worse than the solvent-drop grinding/PXRD benchmark in terms
of the number of the obtained/conrmed cocrystals and (by our
opinion) is much more illustrative. However, if the observation
results are not conrmed by other methods (such as DSC), they
may be misinterpreted. For instance, in the (2-OHBZA + 4-AcA-
mBA) system the thermally unstable cocrystal melts under slow
heating in two stages leading to formation of 4-acetamidobenzoic
acid which can be misinterpreted as an indicator of cocrystal impu-
rity. The differences in cocrystal and eutectic mixture melting pro-
cesses cannot always be easily noticed and cocrystal identication
through repeated melting signicantly increases the experiment
time and makes it similar to that of PXRD.
3.2.3. Saturation temperature method
SMT screening does not require any special analytical equipment
and makes it possible to precisely identify cocrystal formation in a
system. When used on our model systems, STM conrmed the
formation of 4 cocrystals out of 6 possible ones. Since the method
suggests increasing the temperature by DT P 10 C as one of its
conditions, it excludes other side interactions that can lead to a
63
solubility decrease. Therefore, a positive response from STM de-
nitely indicates cocrystal formation.
All cocrystals detected by STM can be obtained by crystalliza-
tion from solution (Rodrguez-Hornedo et al., 2006; Childs et al.,
2008) in analogous conditions which can be further used for
scale-up. The precipitate obtained in the process of using this
method is a pure cocrystal suitable for DSC, IR or PXRD analysis.
However, as in all solvent-based methods, in STM the solvent
should be chosen in advance.
Fig. 9 showing STM results in different solvents indicates that
the higher the ratio of components solubility in a given solvent,
the more difcult it is to identify the cocrystal by this method.
Therefore, it is necessary to screen out from tests the coformers
that are sure to give a negative result if this method is used.
3.2.4. Overall and comparative efciency of the screening methods
General results of screening by different methods are repre-
sented in Table 3.
The number of positive, negative and ambiguous responses for
each of the methods used is shown in Fig. 10.
The represented data show that DSC screening is satisfactorily
accurate and allows screening over a short time without an addi-
tional stage of cocrystal formation. However, high probability of
ambiguous results does not allow it to be used as the only tech-
nique. STM screening, despite its narrow range of application, has
the highest exclusion rate and does not give ambiguous results.
Besides, STM and a combination of DSC and thermal microscopy
methods make it possible to screen out all the systems in which
cocrystal formation from solution and melt is impossible.
The following criteria were used to objectively evaluate the ef-
ciency of the three combined methods: the number of false posi-
tive responses of the method (cases when the method gives a
positive response when no cocrystal is formed), the number of
false negative responses (the methods omission of the systems
with cocrystal formation), the number of systems screened out
by the method and the number of ambiguous results. There are
no false positive responses in any of the three methods, but 2/3
of the systems with negative results are treated by DSC as ambig-
uous. There are false negative responses in thermal microscopy (1
omission out of 6) and STM (2 omissions out of 6) as the cocrystal
under study is unstable under experimental conditions.
The least effective in screening out (though the least labour-
consuming) is the DSC method. Therefore, it can be used at the
Fig. 10. Stacked bar chart representing the ratio of positive (green), uncertain
(yellow) and negative (red) responses for different screening methods used in
present work, in comparison to SDG/PXRD combination. (For interpretation of the
references to colour in this gure legend, the reader is referred to the web version of
this article.).
64 A.N. Manin et al. / European Journal of Pharmaceutical Sciences 65 (2014) 5664
screening early stages for preliminary selection of the leading sys-
tems with the subsequent analysis of ambiguous systems by other
methods such as thermal microscopy and STM.
None of the screening methods was able to denitely identify
cocrystal formation in all the systems when used separately. How-
ever, taking into account the fact that cocrystal formation from
melt is more probable than that in the grinding process (Fucke
et al., 2012), it is possible to use thermal analysis (DSC + thermal
microscopy) as an alternative screening technique to solvent-drop
grinding. STM screening, in its turn, makes it possible both to nd
promising systems and to obtain a stable cocrystal form from
solution.
4. Conclusions
Using combined high-throughput screening methods
signicantly increases the speed and efciency of searching new
pharmaceutical cocrystals without including a stage of cocrystal
formation.
We have evaluated the relative screening efciency of DSC,
thermal microscopy and saturation temperature method, using
solvent-drop grinding/PXRD combination as a benchmark. At an
early stage of research, DSC screening is the most efcient method
as it makes it possible to screen out the systems in which cocrystal
formation is most unlikely before obtaining cocrystals. The method
informative value increases when it is combined with thermal
microscopy which allows conducting a full thermal analysis of
the system.
Theses techniques were used to screen 18 binary systems of
benzoic acid derivatives capable of forming an acid-amide synthon.
As a result, 6 new cocrystals were found and their formation was
proved by PXRD. It was conrmed that the combination of
above-listed techniques is highly efcient in searching for new sol-
uble cocrystals for pharmaceutical industry.
The main advantage of the represented techniques is the possi-
bility to both identify the formation or absence of a cocrystal in a
certain system in the screening process and evaluate the formation
conditions for single crystal growth, which can also be used for
scale-up in industrial production.
Acknowledgments
This work was supported by the Russian Scientic Foundation
(No 14-13-00640). We thank the Upper Volga Region Centre of
Physicochemical Research for technical assistance with DSC and
XRPD experiments.
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