Journal of Molecular Liquids 328 (2021) 115438

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Journal of Molecular Liquids

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

Aqueous solubility and chromatographic studies of antifungal

drug-fluconazole at high temperature conditions
Sema Akay, Berkant Kayan ⁎
Department of Chemistry, Arts and Sciences Faculty, Aksaray University, Aksaray 68100, Turkey

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

Article history: Fluconazole is a novel triazole antifungistatic drug, which can be administered both orally and intravenously and
Received 13 October 2020 is currently used for the treatment of systemic and superficial fungal infections. In this study, the solubility of flu-
Received in revised form 11 January 2021 conazole in water at elevated temperature and pressure was investigated at temperatures in the range of 298 to
Accepted 18 January 2021
473 K under autogenous- 5.0 MPa pressure. The results showed that the solubility of fluconazole was increased
Available online 22 January 2021
146-fold at the highest experimental temperature of 473 K. Based on the experimental data, a mathematical
Keywords:
model was developed to predict the solubility of fluconazole in subcritical water. The model was validated suc-
Solubility cessfully and the theoretical solubility values matched well with the experimental data. Furthermore, a modified
High temperature Apelblat equation provided a good fit to the experimental values except at low temperature. The molar enthalpy
Fluconazole and the molar entropy of dissolution of fluconazole in subcritical water at temperatures ranging from 298 to
Thermodynamic analysis 473 K were calculated. The good solubility of fluconazole in subcritical water allowed us to perform high temper-
Chromatography ature liquid chromatography (HTLC) for the determination of this agent. Moreover, thermogravimetric (TG) and
differential scanning calorimetry (DSC) analysis confirmed that fluconazole had excellent thermal stability under
subcritical conditions.
© 2021 Published by Elsevier B.V.

1. Introduction Due to the higher solubility power compared to ambient tempera-

Thousands of tons of pharmacologically active substances are used
every year to treat diseases and protect public health. The low aqueous
solubility of drugs and their derivatives is one of the main problems
faced by the pharmaceutical industry during the development and test-
ing phase. Subcritical water treatment has appeared in recent years as a
green and efficient method to increase the solubility of drugs in water.
Understanding the dependence of solubility of organics and drugs on
temperature is important for developing subcritical water extraction
and chromatography methods [1–3]. In addition to the subcritical
water extraction and chromatography studies, solubility data in subcrit-
ical water is required to assist in the optimization of various green pro-
cesses, such as environmental remediation, micronization, a medium
for chemistry reactions and decomposition of non-biodegradable poly-
mers. Although many organic compounds have a lot of solubility data
in ambient water, only a few studies have investigated their solubility
in subcritical water. Therefore, obtaining fundamental data such as the
solubility of analytes in high temperature water and analyte stability
are needed to develop green subcritical water technologies and deter-
mine the thermodynamic limit of a process [4–12].
⁎ Corresponding author.
E-mail address: berkantkayan@gmail.com (B. Kayan).
ture water, subcritical water has been extensively used in extraction
processes such as the extraction of low to moderately polar substances
from medicinal plants, which are insoluble in water at ambient condi-
tions. Generally, subcritical water is an effective solvent for the dissoci-
ation and dissolution of a wide range of polar and non-polar compounds
from environmental matrices. Due to its tunable solvating power, it has
also been used in chromatographic separation methods as a mobile
phase [2]. The mechanism behind the tunable solvating power of sub-
critical water mainly involves the reduction of its dielectric constant
and the breaking of the H-bonds as the temperature is increased. It
has been shown that as the temperature is raised, the dielectric constant
of water approaches that of common organic solvents. For instance, at
298 K and 1 atm, the dielectric constant of water is nearly 78.5 whereas
at 473 K and 50 atm, it decreases to 36.63, which is of the same order as
the dielectric constant of acetonitrile (37.5) at 298 K and 1 atm. How-
ever, the temperature dependence of the solubility of various solutes
has been shown to extend beyond the temperature dependence of the
dielectric constant of water [13–16]. In addition to this theory, some re-
searchers showed that the solubility increase is related, instead of the
dielectric constant, to the vaporization or sublimation components of
the solubility of the pure liquid or solid solutes, respectively [17].
Fluconazole [2-(2,4-difluorophenyl)-1,3-bis (1H-1,2,4-tria- zol-1-yl)-
propan-2-ol] is a triazole multifunction antifungal drug applied in
the systemic treatment of fungal infections, such as candidiasis,

https://doi.org/10.1016/j.molliq.2021.115438
0167-7322/© 2021 Published by Elsevier B.V.
S. Akay and B. Kayan
crypto-coccal meningitis, and dermatophytic fungal infections like as-
pergillosis, coccidioidomycosis and histoplasmosis. This drug is in-
cluded in the World Health Organization list of essential medicines
[18]. Its antifungal action is based on the inhibition of the synthesis of
ergosterol, a main ingredient in the fungal cell membrane [19]. Due to
their established use in a wide range of fungal infections, it would be in-
teresting to study fluconazole solubility in different solvents, which can
help the drug find new uses in other diseases [20]. Fluconazole is
slightly soluble in water and its molecular structure is shown in Fig. 1.
However, due to its low aqueous solubility, researchers have focused
on improving its solubility using different methods [21–23]. There is
no experimental or modeling investigation about the solubility of flu-
conazole in subcritical water. This work is the first that deals with the
solubility of fluconazole in subcritical water. First, the solubility of flu-
conazole was assessed at temperatures in the range of 298–473 K and
5.0 MPa pressure, so that water can maintain its liquid state at all tem-
peratures worked. The experimental data were also correlated using a
newly designed approximation model and a modified Apelblat equa-
tion. The solubility and chromatographic behavior of biologically active
substances have been well studied both experimentally and theoreti-
cally, however no such data exist for fluconazole. An additional objec-
tive of this study is to minimize the use of conventional toxic organic
solvents and use high temperature water as the mobile phase.
2. Experimental
2.1. Materials
Fluconazole powder purity of ≥98% (HPLC) was obtained from
Sigma-Aldrich Chemical (Steinheim, Germany). Methanol, acetonitrile
and ortho-phosphoric acid were purchased from Merck Chemical
(Istanbul, Turkey). Ultra-pure water was prepared a MilliPore Milli-Q-
Gradient water purification system (Billerica, MA, USA) with resistivity
of 18.2 MΩ·cm and was used in all experiments. A Zorbax- SB Phenyl
column was purchased from Agilent Technologies (Santa Clara, CA,
USA). A Thermo ODS Hypersil column was acquired from Thermo Fisher
Scientific (Waltham, MA, USA). All reagents were used as received with-
out further modification or treatment and, shown in Table 1.
Fig. 1. Molecular structure of fluconazole.
2
Journal of Molecular Liquids 328 (2021) 115438
Table 1
Provenance and purity of chemicals used in this work.
Chemical name CAS No. Source Mass fraction purity
Fluconazole 86,386–73-4 Sigma-Aldrich >0.990
Methanol 67–56-1 Merck >0.995
Acetonitrile 75–05-8 Merck >0.995
ortho-Phosphoric acid 7664-38-2 Merck >0.995
Water – Our Lab >0.995
2.2. Solubility equipment and methods
Solubility tests were performed in purpose-built equipment, the de-
tailed schematic diagram and methodology of which were fully de-
scribed in our previous work [3,21]. An empty liquid chromatography
column (100 × 4.6 mm id., Thermo ODS Hypersil, Waltham, MA, USA)
was utilized as the equilibration cell. The cell was loaded with 1.0 g of
fluconazole and the end caps were tightened to prevent leakage. A con-
ventional laboratory oven (Teknosem Model column heater system, Is-
tanbul, Turkey) was used to heat the loaded cell at the desired
temperature. An ISCO model 260 D syringe pump (ISCO, Lincoln, NE,
USA) was employed to maintain the flow of distilled water at the cell
at a constant pressure of 5.0 ± 0.1 MPa. This high pressure kept the
water at subcritical conditions and prevented water from boiling inside
the cell. When the selected temperature and pressure reached equilib-
rium, 15 min of static time was allowed before the aqueous phase was
collected. After collection of a 3 mL sample of the heated water-
fluconazole solution, the sample was quickly diluted with methanol to
avoid precipitation and then analyzed by high performance liquid chro-
matography. The collection pipe was also removed and washed with a
methanol-water solution to collect potential residues of fluconazole. Ex-
periments were carried out at eight different temperatures ranging
from 298 to 473 K ± 1 K and in triplicate to ensure reproducibility of
the results.
2.3. High-performance liquid chromatography analysis
An Agilent 1200 HPLC system (Santa Clara, CA, USA) was used for the
chromatographic analysis. It consisted of a binary solvent delivery
pump, an injection valve, a column oven and a diode array detector
set for the detection of fluconazole. For the quantitative determination
of fluconazole, a Zorbax SB Phenyl column (150 × 4.6 mm id) with a
particle size of 5 μm was used. The column temperature was maintained
at 40 ± 1 °C. A mixture of water and methanol at ratio of 70:30 was used
as the mobile phase. The mobile phase was set at isocratic elution mode
and a flow rate of 1.0 mL/min. The volume of the injected sample was
15 μL sample and the fluconazole peak was detected at 260 nm by a
DAD detector.
2.4. Preparation of crystals of subcritical processed fluconazole
Crystals of subcritical processed fluconazole were obtained by dis-
solving fluconazole in subcritical water at 473 K. The aqueous phase
was cooled and crystals formed were harvested by filtration at room
temperature. Then, crystals of subcritical processed fluconazole were
oven-dried for 48 h to use in thermal and spectroscopic analyses.
2.5. TG analysis
In order to ensure that fluconazole was stable in the temperature
ranges for which the solubility data was obtained, the thermal decom-
position was measured by Thermogravimetric analysis (TG). TG was
carried out using a TG (EXSTAR SII TG/DTA 7300) equipped with TG
thermal data analysis software. The sample was weighed in the range
S. Akay and B. Kayan Journal of Molecular Liquids 328 (2021) 115438

2.5–5 mg. The sample was heated from 25 to 650 °C at a heating rate of water's dielectric constant [13–16,23]. The same effect was observed
10 °C min−1 and a nitrogen purge was used. in the case of fluconazole. The values of the mole fraction solubility x2
as a function of temperature are presented in Table 2. When the temper-
2.6. DSC analysis ature was raised from 298 to 473 K, the mole fraction solubility value of
fluconazole increased considerably from 0.08 × 10−3 to 11.73 × 10−3.
The differential scanning calorimetry (DSC) thermograms of flucon- Fig. 2 describes the natural logarithm of the mole fraction solubility
azole were recorded on a Perkin Elmer DSC 400 differential scanning against temperature. There are good correlations between lnx2 and
calorimeter and processed using Perkin Elmer DSC 400 thermal data 1000/T for the models except for Eq. (2) as discussed in the next section.
analysis software (version 11.0). The sample was weighed in the The correlation coefficient is equal to or exceeds 0.999. According to
range 2.5–5 mg and heated from −20 to 400 °C at a heating rate of these experimental results, fluconazole exhibited strong dependence
10 °C min−1 in a crimped sealed aluminum pan, using nitrogen as a of solubility on the temperature of subcritical water with a proportional
purge gas (20 mL/min). increase in solubility as the temperature increased from 323 to 448 K
with a more large solubility increase between 448 and 473 K.
2.7. FT-IR analysis
3.2. Solubility prediction
Fourier transform infrared (FT-IR) spectra of fluconazole were ob-
tained by a Perkin Elmer Spectrum 100 FTIR spectrophotometer (Perkin Eqs. (1) and (2) that follow - developed by Miller et al. to predict the
Elmer, Massachusetts, USA) to characterize the functional groups before solubility of the apolar solid organic compounds such as polycyclic aro-
and after the solubility process at two different temperatures. matic hydrocarbons (PAHs) – were used for the prediction of flucona-
zole solubility in subcritical water [24].
2.8. High-temperature liquid chromatography system

To
ln x2 ðT Þ ¼ ln x2 ðT o Þ ð1Þ
T
A syringe pump and liquid chromatography system were consoli-
dated and designed as a high temperature liquid chromatography sys-

3
To T
tem. Column heating unit was used to temperature control of the ln x2 ðT Þ ¼ ln x2 ðT o Þ þ 15 −1 ð2Þ
T To
separation column and the mobile phase. When reducing the tempera-
ture of the eluent before contact the UV flow cell, a peltier cooling sys- where, x2 is the mole fraction solubility, To is the ambient temperature
tem is connected between separation column and UV detector. The and T is the experimental temperature.
cooling system contain a capillary where is located an aluminum Due to the inadequacy of this equation in estimating the solubility of
block. Perkin-Elmer Chromera® Chromatography UV–Vis dual wave- other organic substances in hot water, researchers gradually modified
length detection system was used for the analysis of fluconazole and de- them and developed new, more accurate mathematical expressions.
tected at 260 nm. A Zorbax SB Phenyl (4.6 × 150 mm, 5 μm) column was Eqs. (3–8) were developed to predict the solubility of alkylbenzenes
used for fluconazole determination using water: acetonitrile: ortho- [25], benzoic and salicylic acids [22], gallic acid, catechin, and
phosphoric acid (89:10:1) mixture as the mobile phase. The flow rate protocatechuic acid [26], parabens [10], paracetamol [27] and 5-
was set between 0.7 and 1.1 mL/min and the injection volume was flurouracil [3] in subcritical water, respectively.
10 μL. Chromatographic analysis was carried out in the temperature


3
range of 373 to 473 K. To T−T o
ln x2 ðT Þ ¼ ln x2 ðT o Þ þ 2 ‐1 ð3Þ
T To
3. Results and discussion

To
ln x2 ðT Þ ¼ 1:85 −1 ln x2 ðT o Þ ð4Þ
3.1. Temperature effect on solubility of fluconazole T



The physicochemical properties of subcritical water are very differ- To To
ln x2 ¼ ln x2 ðT o Þ þ 11 1− ð5Þ
ent in comparison to water at ambient temperature, because the molec- T T
ular kinetic energy of water increases with temperature, and significant


To To
reduction of the intermolecular H-bonding occurs. Since the dielectric ln x2 ¼ ln x2 ðT o Þ þ 0:5ðC−1Þ ‐1 ð6Þ
constant value depends on the extent of H-bonding, it is also reduced T T
as the temperature is raised. It has been established that the solubility


To 8 T−T o
of several hydrophobic compounds in subcritical water increases with ln x2 ¼ ln x2 ðT o Þ þ ð7Þ
T 5 To
temperature and the mechanism is directly related to the decrease of

Table 2
Comparison of mole fraction solubility x2 of fluconazole in subcritical water within the temperature range from T = (298 to 473) K at p = 5.0 MPa.

103 × 2

T/K Exp(x2 ± sd)* Eq. (1) Eq. (2) Eq. (3) Eq. (4) Eq. (5) Eq. (6) Eq. (7) Eq. (8) Eq. (9) Apelblat Eq.

298 0.08 ± 0.01 0.08 0.08 0.01 0.33 0.08 0.08 0.08 0.08 0.24 0.33
323 0.54 ± 0.06 0.17 0.17 0.04 1.29 0.40 0.11 0.19 0.17 0.53 0.63
348 1.27 ± 0.17 0.32 0.34 0.10 4.09 1.53 0.13 0.41 0.32 1.03 1.14
373 1.68 ± 0.23 0.54 0.69 0.23 11.12 4.95 0.16 0.81 0.57 1.85 1.94
398 3.54 ± 0.49 0.87 1.53 0.48 26.63 13.79 0.19 1.49 0.93 3.12 3.18
423 5.40 ± 0.72 1.32 3.99 0.89 57.56 34.03 0.24 2.58 1.45 4.99 5.03
448 7.05 ± 0.83 1.91 12.93 1.49 114.13 75.92 0.26 4.27 2.17 7.64 7.70
473 11.73 ± 1.06 2.66 55.44 2.31 210.52 155.61 0.29 6.80 3.10 11.31 11.50

x2: Based on triplicate measurements mole fraction solubility, sd: Standard deviation of the mean.

3
S. Akay and B. Kayan Journal of Molecular Liquids 328 (2021) 115438

-3 -1
Experimental Eq. 4
-5 R² = 0.9604 -3 R² = 1
ln(x2)

ln(x2)
-7 -5
-9 -7
-11 -9
2 2.5 3 3.5 2 2.5 3 3.5
1000 K/T 1000 K/T

-5
Eq. 1 -1
R² = 1 Eq. 5
-7 -3 R² = 1
ln(x2)

-5

ln(x2)
-9
-7

-11 -9
2 2.5 3 3.5 -11
1000 K/T 2 2.5 3 3.5
1000 K/T

-1
Eq. 2
-3 -7
R² = 0.9314 Eq. 6
-5 R² = 0.9975
ln(x2)

-8
-7
-9 ln(x2)
-9
-11
2 2.5 3 3.5
-10
1000 K/T 2 2.5 3 3.5
1000 K/T

-6 Eq. 3
R² = 0.9988
-3
-8 Eq. 7
ln(x2)

-5 R² = 0.9993
-10
ln(x2)

-7
-12
2 2.5 3 3.5 -9
1000 K/T
-11
2 2.5 3 3.5
Fig. 2. Temperature dependence of fluconazole solubility predicted by different models.
1000 K/T

 
2
To εðT Þ T−T o
ln x2 ¼ ln x2 ðT o Þ þ ð8Þ
T εðT o Þ To Fig. 2 (continued).

An attempt was made to fit the experimental data for fluconazole to

the other approximation models. However, none of them could estimate ambient conditions (Table 2). Therefore, Eq. (9) is a plausible predictor
the solubility of fluconazole with adequate accuracy. The dielectric con- for the solubility of fluconazole in subcritical water.
stant values of water at two different temperatures were used to predict Eq. (10) corresponds to a modified Apelblat equation and was used
the solubility of 5-flurouracil and this modification was tested for the first to correlate the temperature dependency of fluconazole solubility in
time in our previous study [3]. The dielectric constant of water decreases subcritical water [28,29].
as the temperature is raised which subsequently affects the solubility
process. The changed dielectric constant values at different temperatures


are illustrated in Table 3. Therefore, Eq. (9) was modified to include di- To T
ln x2 ¼ A þ B þ C ln ð10Þ
electric constant terms (ε). The mole fraction solubility of fluconazole T To
in subcritical water is described by the following equation:



To εT o The values of A, B, and C were calculated by multilinear regres-
ln x2 ðT Þ ¼ ln x2 ðT o Þ þ ln þ2 ð9Þ
T εT sion. These values were A = −40.2171, B = −615.25 and C =
6.0164. The root-mean-square deviation (RMSD) between the ex-
Validation of Eq. (9) resulted in very accurate fit at higher tempera- perimental and calculated values was determined using the follow-
tures, however it did not describe the solubility process as well near ing Eq. (11):

4
S. Akay and B. Kayan Journal of Molecular Liquids 328 (2021) 115438

-5 Thanks to a pseudochemical reaction process [30], the relationship

Eq. 8
of its dissolution equilibrium constants and activities can be defined
R² = 0.9999
ln(x2) -7 by Eq. (12):
ai
Ki ¼ ð12Þ
-9 as aw

where ai is the activity of fluconazole in the solution, as and aw are the

-11
activities of pure solid, S, and pure liquid, W, respectively.
2 2.5 3 3.5
Owing to standard-state solid and liquid, it is noted that each of as
1000 K/T and aw is considered to be a constant and, equilibrium constants can
be written as below
-3 γi xi
Eq. 9 Ki ¼ ð13Þ
as aw
R² = 0.9997
-5
where γi is the activity coefficient of fluconazole, i, in the solution and xi
ln(x2)

is the mole fraction of fluconazole, i, in the solution.

-7 When taking logarithm of Eq. (13), achievable to Eq. (14).

-9 ln K i ¼ ln xi þ J ð14Þ
2 2.5 3 3.5
where J = ln γi − ln asaw is a temperature-independent constant.
1000 K/T
According to the Gibbs equation and the modified van't Hoff method
[31], the equation for calculating the enthalpies of dissolution ΔsolH can
-3 be accessed using “Apparent thermodynamic analysis”.
Apelblat Eq.
R² = 0.9975 d ln K i
-5 Δsol H ¼ −R −1
ð15Þ
dT
ln(x2)

-7 Substituting the differential of Eq. (14) into Eq. (15) yields.

d ln xi
-9 Δsol H ¼ −R −1
ð16Þ
dT
2 2.5 3 3.5
1000 K/T Using modified Apelblat equation parameters (B and C) to obtain the
differentiation and substituting it into Eq. (16) gives
Fig. 2 (continued).
Δsol H ¼ RT ðC−B=ðT=K ÞÞ ð17Þ
vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
u  2 With knowledge of fundamental thermodynamic relation [32], the
u N
t∑i¼1 xcw,T −xew,T equation for calculating the molar entropies of dissolution ΔsolS can be
RMSD ¼ ð11Þ
N indicated accordingly

Δsol S ¼ RðC−B=ðT=K ÞÞ ð18Þ

where, N represents the number of all values; xcw,T and xew,T represents
the calculated and the experimental solubility in mole fraction.
RMSD was determined as 0.000338. The theoretical solubility values
of fluconazole, as calculated by the modified Apelblat equation,
matched well to the experimental values at higher temperatures, as
shown in Table 2. At lower temperatures, experimental and calculated
values do not match well and the fact that the numbers are small in-
creases the error considerably. Overall, the Apelblat equation is suitable
for the correlation of theoretical and experimental solubility values of
fluconazole in water. The discrepancies at low temperature do not affect
the applicability of the model significantly.
Modified Apelblat equation is used to determine the molar enthalpy
and molar entropy of dissolutions (ΔsolH and ΔsolS) of solid compounds.
According to Table 3, it is indicated that solubility of fluconazole in sub-
critical water in the experimental temperature range was endothermic,
ΔsolH > 0, and ΔsolS for fluconazole dissolving in subcritical water was rela-
tively large. The positive ΔsolH and ΔsolS for this process elicited that flucon-
azole being dissolved in subcritical water was an entropy-driving process.
The enthalpic component ΔsolH has a positive value at every temper-
ature studied, that is, the dissolution of fluconazole in subcritical water is
endothermic. The ΔsolH shows a marked increase with increasing tem-
perature from 20.05 kJ·mol−1 at 298 K to 28.77 kJ·mol−1 at 473 K,
where the isobaric heat capacity of solution ΔCp,sol defined by Eq. (19):


∂ΔH sol
ΔC p,sol ¼ ¼ RC ð19Þ
∂T p

Table 3 is 50.02 J·mol−1·K−1. The large positive ΔCp,sol causes the significant in-
Apparent thermodynamic properties of dissolution process of FLC in subcritical water at crease in the solubility of fluconazole in subcritical water with increas-
the temperature range from T = (298 to 473) K and dielectric constant of water at
ing temperature [33]. Some studies are indicated that the cause of
p = 5.0 MPa [24].
positive entropy during the dissolution process is that solute disrupted
T/K 298 323 348 373 398 423 448 473 the alignment of solvent molecule and therefore decreased the degree
ΔsolH(kJ/mol) 20.05 21.26 22.52 23.77 25.02 26.27 27.52 28.77 of order of the system while it was dissolved in solvent [34,35].
ΔsolS(J/mol.K) 67.18 65.80 64.71 63.73 62.87 62.11 61.43 60.83 The endothermic effect in the dissolving process (ΔsolH > 0) may be
ΔCp,sol(J/mol.K) 50.02 50.02 50.02 50.02 50.02 50.02 50.02 50.02 explain possibly thanks to the interactions between fluconazole mole-
Єr 78.65 70.09 62.42 55.59 49.52 44.12 39.28 34.90
cules and water molecules are more powerful than those between the

5
S. Akay and B. Kayan Journal of Molecular Liquids 328 (2021) 115438

Fig. 3. TG thermograms of (a) raw fluconazole (298 K) b) subcritical processed fluconazole (473 K).

6
S. Akay and B. Kayan
water-water molecules. Due to containing the –F, –OH and –N= of flu-
conazole molecule, fluconazole may have different forces such as elec-
trostatic force, hydrogen bond, partial hydrophobic interaction in the
solubility process. Nevertheless, hydrogen bonding strength the be-
tween of molecules are considered as the most reasonable approach in
subcritical water process. Hydrogen bonds in water are spontaneously
related because the strength of a hydrogen bond is related by the pres-
ence of other hydrogen bonds nearby. Hence a little alteration to one
hydrogen bond affects the whole water volume. Changes in hydrogen
bonding strength affect the dielectric constant and heat of vaporization
values. The dielectric constant value is higher because of stronger hy-
drogen bonds at lower temperatures. When the increasing temperature
of the water, the raised thermal swash agitation decreases the strength
of each hydrogen bond and causes to a evident reduction in dielectric
constant value. The solubility of poorly soluble substances in water gen-
erally increases, as the polarity of the water molecules decreases de-
pending on the weakness of the hydrogen bond strength [13,36–38].
3.3. Thermal analysis and stability
3.3.1. TG thermograms
To understand whether fluconazole was degraded under subcritical
conditions, the TG thermogram of fluconazole obtained after the solu-
bility study was examined (Fig. 3).
Fig. 4. DSC thermograms of (a) raw fluconazole (298 K) b) subcritical processed fluconazole (473 K).
7
Journal of Molecular Liquids 328 (2021) 115438
A TG was record on a sample of fluconazole dissolved at 473 K and
dried according to the experimental method. Fig. 3a corresponds to
the thermal behavior of the raw fluconazole sample (298 K) and
Fig. 3b was obtained from subcritical processed fluconazole (473 K)
after the solubility experiment at 473 K. Fig. 3a and b show that there
is no mass loss in the temperature range 100–200 °C before melting oc-
curs. However, it is possible that the presence of water in the product re-
sulted in a reduced melt peak temperature. A mass loss is observed in
the maximum peak at the temperature range of 200–355 °C. Fig. 3b
shows 96.3% mass loss in the temperature range 200–355 °C in TG
curve and this mass loss occurred in the temperature range correspond-
ing to degradation of fluconazole. At the end of the analysis, it was de-
termined that approximately 4% of the substance remained unbroken.
As a result, it was stated that the decomposition temperature for
fluconazole was at 200–355 °C, i.e. above the working temperature,
and therefore thermal decomposition did not influence the solubility
measurements.
3.3.2. DSC thermograms
DSC is a very advisable method to study the thermal stability behav-
ior of pharmaceutical substances at different temperatures. It is very
sensitive to phase transitions that are associated with changes in the
heat capacity of the sample. DSC thermograms were used to analyze
and characterize the raw and subcritical processed fluconazole. Fig. 4
S. Akay and B. Kayan Journal of Molecular Liquids 328 (2021) 115438

shows DSC thermograms of raw and subcritical processed fluconazole.

The stability of fluconazole was determined constant up to maximum
operating temperature of 473 K in subcritical conditions.
Fig. 4a corresponds to the thermal behavior of the raw fluconazole
sample (298 K) and fluconazole exhibited three exothermic peaks at
102.27, 143.67 and 304.53 °C. The first peak corresponds to dehydration
of the crystal, and the temperature agrees with the boiling point of
water. The second exothermic peak corresponds to the melting point
of the crystals. The third peak corresponds to degradation of fluconazole
as shown in TG thermograms.
Fig. 4b was obtained from subcritical processed fluconazole after the
solubility experiment at 473 K. Three exothermic peaks are seen at
103.54, 142.44 and 302.24 °C. The processed fluconazole DSC curve in-
dicated a peak melting temperature that was 1.27 °C less than the
peak melting temperature of the raw material. The onset of melting oc-
curred at nearby the 140 °C for both samples. Considering that the onset
of melting is identical for the raw and SBCW processed fluconazole sam-
ples, it can be concluded that the processed fluconazole has the same
crystal structure as the raw material. It is possible that the reduced melt-
ing peak for the SBCW-processed fluconazole was due to trace amounts
of water left in the product matrix post-processing. The small differ- Fig. 6. High temperature liquid chromatograms of fluconazole (a) and Fluzole® (b) at
ences in the thermograms in Fig. 4a-b may be caused by impurities in different temperatures.
fluconazole. As a result, fluconazole was found to exhibit similar ther-
mal behavior of fluconazole monohydrate as reported in the literature
by Alkhamis et al., [39] and stable at the temperatures studied. oxidizing capacity of water was practically eliminated. However, even
if a low oxygen concentration still remained dissolved after degassing,
3.3.3. FT-IR spectra the high fluconazole concentration used meant that any potential flu-
The comparative FTIR results provided an insight to potential conazole degradation went unnoticed.
changes of fluconazole functional groups. The spectra obtained for flu-
conazole samples before (298 K) and after (473 K) the solubility process 3.4. High temperature liquid chromatographic analysis
showed almost identical peaks (Fig. 5). Therefore, it can be concluded
that fluconazole was thermally stable in subcritical water conditions. Determination of fluconazole has been investigated using high tem-
The remarkable stability of fluconazole in these conditions may be due perature liquid chromatography, known as green chromatography
to the water-degassing step that preceded the solubility investigation. techniques. Fluconazole determination was performed at a temperature
Through this step, most of dissolved oxygen was removed and the of 373 K and the eluent consisted of water:acetonitrile:ortho-

Fig. 5. FT-IR spectra of untreated fluconazole (298 K) and after the solubility test (473 K).

8
S. Akay and B. Kayan Journal of Molecular Liquids 328 (2021) 115438

Table 4 Appendix A. Supplementary data

Temperature effect on high-temperature liquid chromatographic determination of flucon-
azole and Fluzole®.
Supplementary data to this article can be found online at https://doi.
T (K) Flow rate Retention Capacity Asymmetry Peak width Plate org/10.1016/j.molliq.2021.115438.
(mL/min) time (min) factor factor (W0.5) Number

FLC References
373 0.7 8.09 31.35 0.97 0.73 680
398 0.8 5.85 22.40 0.97 0.51 729 [1] E. Zuccato, D. Calamari, M. Natangelo, R. Fanelli, Presence of therapeutic drugs in the
423 0.9 4.50 17.00 0.99 0.38 777 environment, Lancet 355 (2000) 1789.
448 1.0 3.62 13.47 0.98 0.30 807 [2] Y. Pu, F. Cai, D. Wang, Y. Li, X. Chen, A.G. Maimouna, Z. Wu, X. Wen, J.-F. Chen, N.R.
473 1.1 3.18 11.73 1.00 0.25 896 Foster, Solubility of bicalutamide, megestrol acetate, prednisolone, beclomethasone
Fluzole® dipropionate, and clarithromycin in subcritical water at different temperatures from
383.15 to 443.15 K, J. Chem. Eng. Data 62 (2017) 1139.
373 0.7 8.17 31.68 0.94 0.80 578
[3] S. Akay, B. Kayan, Y. Yang, Solubility and chromatographic separation of 5-
398 0.8 5.92 22.64 0.95 0.62 505
fluorouracil under subcritical water conditions, J. Chem. Eng. Data 62 (2017) 1538.
423 0.9 4.61 17.18 0.96 0.53 419
[4] Y. Yang, S. Bowadt, S.B. Hawthorne, D.J. Miller, Subcritical water extraction of
448 1.0 3.70 13.69 0.96 0.44 392
polychlorinated biphenyls from soil and sediment, Anal. Chem. 67 (1995) 4571.
473 1.1 3.25 11.90 0.97 0.40 366
[5] R.M. Smith, Extractions with superheated water, J. Chromatogr. A 975 (2002) 31.
[6] J. Kronholm, P. Vastamäki, R. Räsänen, A. Ahonen, K. Hartonen, M.L. Riekkola, Ther-
mal field-flow fractionation and gas chromatography−mass spectrometry in deter-
mination of decomposition products of expandable polystyrene after reactions in
pressurized hot water and supercritical water, Ind. Eng. Chem. Res. 45 (2006) 3029.
phosphoric acid (89:10:1). As illustrated in Fig. 6, the determination of [7] E.S. Ong, J.S.H. Cheong, D. Goh, Pressurized hot water extraction of bioactive or
fluconazole and Fluzole® were successfully achieved by high tempera- marker compounds in botanicals and medicinal plant materials, J. Chromatogr. A
ture liquid chromatography at all temperatures. It is worth noting that 1112 (2006) 92.
[8] A.G. Carr, R. Mammucari, N.R. Foster, Solubility and micronization of griseofulvin in
as the temperature was raised, the retention time of fluconazole de- subcritical water, Ind. Eng. Chem. Res. 49 (2010) 3403.
creased. This was mainly due to the reduced column backpressure at el- [9] M.S.S. Curren, J.W. King, Solubility of triazine pesticides in pure and modified sub-
evated temperatures, as a result of lower eluent velocity and higher critical water, Anal. Chem. 73 (2001) 740.
solute diffusivity. The HTLC chromatogram of the Fluzole® tablet [10] B. Kapalavavi, J. Ankney, M. Baucom, Y. Yang, Solubility of parabens in subcritical
water, J. Chem. Eng. Data 59 (2014) 912.
(Fig. 6(b)) was essentially the same with respect to the number of [11] W.H. Teoh, Fundamental Solubility Study of Polycyclic Aromatic Hydrocarbons in
peaks, peak intensity, retention times and order. No extra peak or any Subcritical Water and Ethanol Mixtures, Doctor of Philosophy Thesis The University
degradation product was observed during the elution. Table 4 presents of New South Wales, Australia, 2012.
[12] Z. Knez, D. Cör, M.K. Hrncic, Solubility of solids in sub- and supercritical fluids: a re-
the effect of temperature on the chromatographic determination of flu- view 2010–2017, J. Chem. Eng. Data 63 (2018) 860.
conazole and Fluzole®. The use of high temperatures also improved the [13] A.G. Carr, R. Mammucari, N.R. Foster, A review of subcritical water as a solvent and
mass transfer within the chromatographic column. The fluconazole its utilisation for the processing of hydrophobic organic compounds, Chem. Eng. J.
peak is sharp and symmetrical at all temperatures. Overall, a tempera- 172 (2011) 1.
[14] I. Nezbeda, J. Pavlicek, Application of primitive models of association: a simple the-
ture raise of 100 K (from 373 to 473 K) combined with a higher flow oretical equation of state of water, Fluid Phase Equilib. 116 (1996) 530.
rate of 1.1 mL/min, lead to the reduction of retention time from 8.09 [15] M. Nakahara, N. Matubayasi, C. Wakai, Y. Tsujino, Structure and dynamics of water:
to 3.18 min. from ambient to supercritical, J. Mol. Liq. 90 (2001) 75.
[16] E.R. Caffarena, J.R. Grigera, On the hydrogen bond structure of water at different
densities, Phys. A (Amsterdam, Neth.) 342 (2004) 34.
4. Conclusions [17] N. Galamba, A. Paiva, S. Barreiros, P. Simões, Solubility of polar and nonpolar aro-
matic molecules in subcritical water: the role of the dielectric constant, J. Chem.
The aqueous solubility of antifungal drug fluconazole was investi- Theory Comput. 15 (2019) 6277.
[18] P.A. Basford, K.R. Back, M. Cram, R. Docherty, R.J. Davey, A.J. Cruz-Cabeza, Impact of
gated in the temperature range of 298 to 473 K at subcritical water con- crystal structure and molecular conformation on the hydration kinetics of flucona-
ditions. The direct and clear dependency of solubility on temperature zole, Cryst. Growth Des. 19 (2019) 7193.
was established. It was observed that raising the temperature from [19] B.C.D. Owoyemi, C.C.P. Silva, M.S. Souza, L.F. Diniz, J. Ellena, R.L. Carneiro, Flucona-
zole: synthesis and structural characterization of four new pharmaceutical cocrystal
298 to 473 K resulted in 146-fold increase of solubility. The apparent
forms, Cryst. Growth Des. 19 (2019) 648.
thermodynamic analysis results showed that enthalpy and entropy [20] K. Bhesaniya, K. Nandha, S. Baluja, Thermodynamics of fluconazole solubility in var-
values were positive, thus the dissolution process was endothermic ious solvents at different temperatures, J. Chem. Eng. Data 59 (2014) 649.
and entropy-driving process. A new solubility model was developed to [21] S. Akay, B. Kayan, D. Cunbin, J. Wang, Y. Yang, Research of sulfadiazine using subcrit-
ical water and water+alcohol mixtures as the solvent: solubility and thermody-
estimate the solubility of fluconazole at high temperature conditions namic property, J. Mol. Liq. 253 (2018) 270.
due to the inadequacy of earlier models. The newly developed model [22] B. Kayan, Y. Yang, E.J. Lindquist, A.M. Gizir, Solubility of benzoic and salicylic acids in
produced theoretical values comparable with the experimental ones. subcritical water at temperatures ranging from (298 to 473) K, J. Chem. Eng. Data 55
(2010) 2229.
An Apelblat equation was applied and compared with experimental
[23] M. Uematsu, E.U. Franck, Static dielectric constant of water and steam, J. Phys. Chem.
values and provides a good match except at low temperatures. Thermal Ref. Data 9 (1980) 1291.
analysis methods (TG/DSC) were used to analyses the behavior of flu- [24] D.J. Miller, S.B. Hawthorne, A.M. Gizir, A.A. Clifford, Solubility of polyacyclic aromatic
conazole in the process of subcritical process. Subsequently, determina- hydrocarbons in subcritical water from 298 to 498 K, J. Chem. Eng. Data 43 (1998)
1043.
tion of fluconazole was successfully achieved in high temperature liquid [25] J. Mathis, A.M. Gizir, Y. Yang, Solubility of alkylbenzenes and a model for predicting
chromatography. the solubility of liquid organics in high-temperature water, J. Chem. Eng. Data 49
(2004) 1269.
Declaration of Competing Interest [26] K. Srinivas, J.W. King, L.R. Howard, J.K. Monrad, Solubility of gallic acid, catechin, and
protocatechuic acid in subcritical water from (298.75 to 415.85) K, J. Chem. Eng.
Data 55 (2010) 3101.
The authors declare that they have no known competing financial [27] Z. Emire, E. Yabalak, A.M. Gizir, Solubility and degradation of paracetamol in subcrit-
interests or personal relationships that could have appeared to influ- ical water, J. Serb. Chem. Soc. 81 (2016) 1.
[28] A. Apelblat, E. Manzurola, Solubilities of o-acetylsalicylic, 4-aminosalicylic, 3,5-
ence the work reported in this paper. dinitrosalicylic, and p-toluic acid, and magnesium-DL-aspartate in water from T =
(278 to 348) K, J. Chem. Thermodyn. 31 (1999) 85.
Acknowledgements [29] A. Apelblat, E. Manzurola, Solubilities of L-glutamic acid, 3-nitrobenzoic acid,
p-toluic acid, calcium-L-lactate, calcium gluconate, magnesium-DL aspartate, and
magnesium-L-lactate in water, J. Chem. Thermodyn. 34 (2002) 1127.
The authors gratefully thank Scientific Research Project Unit (BAP) [30] F.A. Wang, Molecular Thermodynamics and Chromatographic Retention, Meteorol-
of Aksaray University (2019-004) for the financial support of this work. ogy Press, Beijing, 2001.

9
S. Akay and B. Kayan
[31] D. Bourgois, D. Thomas, J.L. Fanlo, J. Vanderschuren, Solubilities at high dilution of
toluene, ethylbenzene, 1, 2, 4-trimethylbenzene, and hexane in di-2-ethylhexyl,
diisoheptyl, and diisononyl phthalates, J. Chem. Eng. Data 51 (2006) 1212. [36]
[32] J.M. Prausnitz, R.N. Lichtenthaler, E.G. Azevedo, Molecular Thermodynamics of
Fluid-Phase Equilibria, Prentice Hall PTR, New Jersey, 1999. [37]
[33] Y. Takebayashi, K. Sue, S. Yoda, Y. Hakuta, T. Furuya, Solubility of terephthalic acid in
subcritical water, J. Chem. Eng. Data 57 (2012) 1810.
[34] C-L. Zhang, F. Zhao, Y. Wang, Thermodynamics of the solubility of sulfamethazine in [38]
methanol, ethanol, 1-propanol, acetone, and chloroform from 293.15 to 333.15 K J.
Mol. Liq. 159 (2011) 170. [39]
[35] S. Suren, N. Sunsandee, M. Stolcova, M. Hronec, N. Leepipatpiboon, P. Ura, S.
Kheawhom, Measurement on the solubility of adipic acid in various solvents at
10
Journal of Molecular Liquids 328 (2021) 115438
high temperature and its thermodynamics parameters, Fluid Phase Equilib. 360
(2013) 332.
S.R. Allada, Solubility parameters of supercritical fluids, Ind. Eng. Chem. Process. Des.
Dev. 23 (1984) 344.
H. Ohtaki, T. Radnai, T. Yamaguchi, Structure of water under subcritical and super-
critical conditions studied by solution X-ray diffraction, Chem. Soc. Rev. 26 (1997)
41.
R.J. Fernández-Prini, H.R. Corti, M.L. Japas, High-Temperature Aqueous Solutions:
Thermodynamic Properties, CRC Press, Boca Raton, 1991.
K.A. Alkhamis, A.A. Obaidat, A.F. Nuseirat, Solid-state characterization of fluconazole,
Pharm. Dev. Technol. 7 (2002) 491.