Journal Pre-proofs

Evaluation of water induced phase transition of Fexofenadine Hydrochloride

during wet granulation process using NIR and DSC techniques

Suye Li, Yanna Zhao, Lili Wang, Hengqian Wu, Yan Gao, Lingxuan Zhang,
Zhengping Wang, Jun Han

PII: S0026-265X(21)00581-6
DOI: https://doi.org/10.1016/j.microc.2021.106497
Reference: MICROC 106497

To appear in: Microchemical Journal

Received Date: 6 April 2021

Revised Date: 31 May 2021
Accepted Date: 1 June 2021

Please cite this article as: S. Li, Y. Zhao, L. Wang, H. Wu, Y. Gao, L. Zhang, Z. Wang, J. Han, Evaluation of
water induced phase transition of Fexofenadine Hydrochloride during wet granulation process using NIR and
DSC techniques, Microchemical Journal (2021), doi: https://doi.org/10.1016/j.microc.2021.106497

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 Evaluation of water induced phase transition of Fexofenadine

Hydrochloride during wet granulation process using NIR and DSC

techniques

Suye Lia, Yanna Zhaob,*, Lili Wangc, Hengqian Wuc, Yan Gaod, Lingxuan Zhangd,
Zhengping Wangc,d, Jun Hana,b,c,d,*
aCollege of Chemistry, Chemical Engineering and Materials Science, Shandong
Normal University, Jinan 250014, People’s Republic of China
bInstitute of BioPharmaceutical Research, Liaocheng University, Liaocheng,
Shandong, 252000, People’s Republic of China
cSchool of Chemistry and Chemical Engineering, University of Jinan, Jinan 250022,
People’s Republic of China
dLiaocheng High-Tech Biotechnology Co. Ltd., Liaocheng, Shandong, 252000,
People’s Republic of China
Corresponding author information
*Jun Han, PhD
Tel: +86 0635 8239136
E-mail address: junhanmail@163.com
*Yanna Zhao, PhD
Tel: +86 0635 8239345
E-mail address: ynzhao2011@163.com

1
Graphical Abstract

2
Abstract
The quality control of drug products during manufacturing processes is important,
particularly the presence of different polymorphic forms in active pharmaceutical
ingredients (API) during production, which could affect the performance of the
formulated products. In this study, Fexofenadine Hydrochloride (FXD) Form I
(anhydrous) was used as the starting material for pharmaceutical formulation, while
FXD exhibited a polymorphic conversion to hydrate Form II in presence of water. It is
necessary to monitor phase transition from Form I to Form II during wet granulation.
The aim of this paper was to quantify the polymorphic forms of FXD during wet
granulation process by NIR and DSC technique. Calibration models were developed
and validated in binary mixtures of FXD polymorphs forms and multicomponent
mixtures of FXD formulation. The NIR and DSC can be successfully employed to
evaluate and quantify Form I during wet granulation. The Form I % determined was
similar by NIR and DSC. The relationship between phase transition of FXD and water
content was determined. In addition, the effect of various excipients on the phase
transition of FXD was also evaluated by NIR. The understanding of the phase transition
in wet granulation process of pure FXD, formulation and FXD-excipient mixtures
provided directions for optimization of FXD manufacturing processes.
Keywords: Fexofenadine Hydrochloride; Polymorphic characterization; Wet
granulation; Phase transition; NIR spectroscopy; DSC

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1. Introduction
Polymorphism widely exists in solid matter, especially in pharmaceuticals.
Polymorphism is essentially identical in chemical composition but different in crystal
conformation, which would impact the physical, chemical, and mechanical properties,
including the solubility, chemical stability, hygroscopicity, particle size and shape,
flowability and mechanical behaviors, etc [1, 2]. Those undesired changes caused by
polymorphism potentially occurred during drug development and formulation which
might affect the product quality and performance, drug product processability and
manufacturability [3, 4]. Even unexpected appearance or disappearance of a
polymorphic form may lead to the bioavailability and therapeutic efficacy difference of
the active pharmaceutical ingredients (APIs) [5]. To anticipate and prevent such
product inconsistencies, it is vital to fully identify the different polymorphs, and
monitor a polymorphic occurrence in the API or excipients. Furthermore, it is crucial
to proper select and control excipients and to assess the effect of manufacturing
processes and processing conditions on polymorphic conversions [6].
“Many analytical techniques are able to detect solid-state characterization for
quantitative and qualitative analysis such as near infrared (NIR), Raman spectroscopy,
powder X-ray diffractometry (PXRD), and thermal methods have been used for
quantitative estimation of polymorphic forms of drugs [7-10]. NIR spectroscopy
technology is widely developed as a common method for pharmaceutical applications
due to its inherent characteristics including non-destruction, reagent-free in the process,
fast operation and easy applicability for quality monitoring together with control
processes of pharmaceutical products [7, 11-13]. The application range of NIR and
Raman has been widespread in the recent years [8, 14, 15]. The range of application of
NIR and Raman overlap greatly, for example, in identification and quantification of
raw materials. Several comparative studies on the quantification of polymorphs forms
using NIR and Raman have been reported [16]. César Pino-Torres et.al concluded that
both NIR and Raman techniques delivered adequate precision results for the quality
analysis control of a solid pharmaceutical formulation with three active ingredients [7].
Valmala Bhavana et.al described that NIR and Raman were equivalent in % NHa
prediction for granulation technique [9]. PXRD tends to be applied as qualitative
analysis approach due to the issues of preferred orientation and interference from
crystalline excipients [10, 17]. Differential scanning calorimetry (DSC) is a very well-

4
established technique for pharmaceuticals field like understanding of phase behavior of
aqueous carbohydrate systems [18], quantification of the extent of phase separation [19],
estimation of mixing uniformity [20], quantification of the polymorphs [21, 22] etc. In
recent study, DSC assisted with dissolution test was even utilized to evaluate the quality
of drug tablets [23]. Generally, all technologies have their own advantages in terms of
quantitative analysis application. In this paper, during wet granulation process, water-
induced phase transition of Fexofenadine Hydrochloride was evaluated by utilizing
NIR combined with another well-established technique DSC.
Fexofenadine Hydrochloride (FXD, Figure 1) is a white to off-white crystalline
powder and a third-generation antihistamine drug, which mechanism of action results
from a competitive and reversible inhibition of the H1 receptor [24]. It is marketed under
the trade name ALLEGRA, which is one of the blockbusters in the section of
antihistamines to treat allergic coryza and idiopathic urticaria and similar disease [25].
Lokesh Kumar et al. has given an introduction on the properties and polymorphism of
Fexofenadine Hydrochloride [26]. It is disclosed that only FXD form I (anhydrous) is
used as the starting material in the commercial pharmaceutical formulation [27].
Furthermore, FXD is sensitive to solvent so that the presence of water could provide
the chance of partially converting anhydrous Form I into hydrous Form II. Additionally,
it is important to realize that only the fraction of drug that exposure to water may occur
of undergoing transformation. Based on the understanding of quality by design (QbD),
it was necessary to investigate the physical or chemical interactions between APIs and
excipients in FXD manufacturing process. Thus, the aim of the study was to utilize the
NIR and DSC technologies to quantify the polymorphic forms of FXD during wet
granulation process. Meanwhile, the influence of excipient on the phase transition of
FXD in wet granulation was investigate and quantify by NIR spectroscopy. The
ultimate significance of the study is to guide the proper selection of process parameters.
2. Experimental section
2. 1 Materials
Fexofenadine Hydrochloride (FXD, C32H40ClNO4), was purchased from Ind-
Swift Laboratories Limited (India, Lot # H007049003) and used without further
purification. The ultrapure water was prepared by Milli-Q® Advantage A10® water
purification system (Millipore, USA, electrical resistivity was 18.2 MΩ·cm at 25 °C).
And the following were used as excipients: Pregelatinized starch (Colorcon, USA, Lot

5
# IN539042), Microcrystalline cellulose (FMC Biopolymer, USA, Lot # P115828088),
and Croscarmellose sodium (FMC Biopolymer, USA, Lot # TN16829944).
2. 2 Methods
2. 2. 1 Preparation of Form II
Fexofenadine Hydrochloride Form I is the starting material used in the
pharmaceutical formulation. Form II can be generated in presence of water. Thus, the
pure Form II was prepared via the wet granulation process of FXD alone. Further drying
was carried out under electro-thermostatic blast oven at 60 °C for 24 h. And the
characterization of Fexofenadine Hydrochloride Form II was analyzed by infrared
spectroscopy (IR) and powder X-ray diffraction (PXRD).
2. 2. 2 Instrumentation
2. 2. 2. 1 PXRD
PXRD patterns were obtained by Ultima IV X-ray diffractometer (Rigaku, Japan)
with a CuKα radiation (1.541836 Å) at room temperature. The tube voltage and current
were set at 40 kV and 40 mA, respectively. The divergence slit and anti-scattering slit
settings were set at 0.5˚ for the illumination on the 10 mm sample size. Sample was
measured by a continuous scan between 3˚ and 40˚ in 2θ with a step size of 0.02˚.
2. 2. 2. 2 FTIR
Infrared (IR) spectrum was collected by a Nicolet 6700 Fourier-Transform
Infrared Spectroscopy (FT-IR) spectrometer (Thermo Scientific, USA) using
Potassium Bromide pellets technique in the region 500 – 4000 cm-1.
2. 2. 2. 3 DVS measurements
Vapor sorption isotherm was performed on a Dynamic Vapor Sorption advantage
instrument (Surface Measurement System Ltd., London, UK) under a 200 mL stream
of N2 at 25 °C. The samples (~ 30 mg) were mounted on a balance, and were studied at
a humidity range from 0 to 90% with 10% stepwise. The measurement method type
was taken with dm/dt 0.002%/min on a minimum stage time of 5 min (maximum stage
time of 360 min and stability duration of 10 min), and the further steps were controlled
automatically.
2. 2. 3 Preparation of calibration and validation samples
2. 2. 3. 1 Binary mixtures of FXD polymorphs forms
200 mg standard mixtures of the FXD Form I and Form II polymorphs containing
0%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% and 100% FXD Form I were

6
weighted by an XPE205DR analytical balance (Mettler Toledo, USA) and properly
mixed. To prepare homogenized mixtures, all the components were kept in self-sealing
bags and mixed by manual shaking for 100 times in longitudinal and transverse
directions to provide uniformity [28]. Then, about 100 mg homogenized powder was
kept in glass vials until NIR and DSC analysis. The validation samples containing Form
I levels of 10%, 50%, and 80% were prepared to verify the accuracy between the value
calculated by models and actual value.
2. 2. 3. 2 Multicomponent mixtures of FXD formulation
The formula of the core tablet contains 30 mg Fexofenadine Hydrochloride and
the following excipient: croscarmellose sodium, microcrystalline cellulose, magnesium
stearate, and pregelatinized starch. Based on the pharmaceutical composition reported
in patent US20030203020A1, the compositions for wet granulation include
Fexofenadine Hydrochloride (API), pregelatinized starch, microcrystalline cellulose,
and croscarmellose sodium presented 40:40:17:3 of mass fraction range [25, 29]. About
200 mg of multicomponent mixtures were prepared by weighing 80 mg FXD
polymorphs mixtures in a similar Form I concentration range as the previous ones with
120 mg constant amounts of formula excipients. The FXD and excipients were
previously passed through 60 mesh sieve to control the differences in particle size. The
multicomponent samples were thoroughly mixed in similar fashion as the previous ones.
The validation samples of multicomponent mixtures containing 10%, 50%, and 80%
Form I were also prepared.
2. 2. 3. 3 Preparation of excipient-FXD polymorphs forms mixtures
In order to thoroughly understand and investigate the effect of main excipients on
phase transition of FXD in wet granulation, the NIR quantitative model of %Form I in
pregelatinized starch-FXD polymorphs and microcrystalline cellulose-FXD polymorph
mixtures was development. Calibration samples were prepared by mixing 100 mg
polymorphs mixtures with Form I in concentration range from 0% to 100%, with single
excipient (pregelatinized starch/ microcrystalline cellulose) to reach a total 200 mg of
excipient-FXD polymorphs forms ternary mixtures. The samples were mixed as evenly
as possible and kept in glass vials till sample analysis by NIR. Validation samples
containing 10%, 50%, and 80% Form I were prepared.
2. 2. 3. 4 NIR quantitative method development
Fourier transformation near infrared spectrometer were performed on NIRFlex N-

7
500 spectrometer (Büchi. Swiss). All NIR spectra were measured by accumulating 32
scans at wave number range from 4000 cm−1 to 10,000 cm−1 with 4 cm−1 spectral
resolution. Data was acquired using NIRWare software suite. The best model was
selected based on conventional criteria, Q value, coefficient of determination (R2), root-
mean-square error of calibration values (RMSEC), root-mean-square error of
predictions values (RMSEP), and the standard error of estimation, SEE (referred to as
SEC when calculated for the C-set and SEP for the V-set).
2. 2. 3. 5 DSC quantitative method development
Differential scanning calorimetry was conducted using Discovery DSC (TA
Instruments, USA). The instrument was calibrated with indium standard. The sample
(2 – 5 mg) was weighted by an analytical balance (MSA6-6S, Satorius, ± 0.001 mg),
then placed in pinhole aluminum DSC pan and scanned at ramping rate of 10 °C/min
under a dry nitrogen atmosphere. The heating ramp of DSC curves were as follow:
Ramp 1 = heating rate of 10 °C /min from 30 °C to 220 °C.
Several authors attempted to establish a linear relation with enthalpy value for
quantitative analysis [23, 30]. For this method, the accuracy of the enthalpy
determination is important. The commonly used linear base line results in systematic
errors of the enthalpy determination by peak integration [18]. Important for the enthalpy
determination is the selection of the integration limits. In this study, the following
parameters were analyzed from the melting curve (Figure 2). The following
experiments were used this sigmoidal baseline type. The obtained data was plotted for
FXD Form I % vs. enthalpy of drug to get a linear calibration graph.
2. 2. 3. 6 Accuracy and precision of quantitative methods
The NIR and DSC quantitative methods were validated for precision and accuracy.
The accuracy was evaluated by the recovery of the validation samples of above known
concentration (10%, 50% and 80% FXD Form I). The accuracy was expressed as
recovery value.
Recovery (R): R = Xcalculated / Xactual * 100%
Xcalculated: % FXD Form calculated or predicted by Quantitative methods
Xactual: %FXD Form I actual value
The precision was evaluated by repeatability. For NIR and DSC quantitative
methods, the repeatability were studied by measuring 50% Form I concentration six
times on different days. The result was expressed as relative standard deviation (% RSD)

8
calculated using the following equation. SD and Xaverage represented the standard
deviation and average of the predicted value of the sample.
%RSD = SD/ Xaverage *100
2. 2. 4 Application of the models
2. 2. 4. 1 Apparatus
Wet granulation experiments were carried out in high shear granulators (FHSG
20, Foryou Mechatronics LTD.) with 2 L stainless steel bowl. The high shear granulator
was equipped with spray system (Spraying Systems Co.) including spray nozzles and
pressure tank to make the water stay spray pattern and spray evenly on the materials
(Figure S1). The pressure of the spraying system was 0.3 MPa. A pre-calculated amount
of water was added by spraying into the granulator. The final water content of the
samples after wet granulation was measured by Karl Fischer Titration (851 Titrando,
Metrohm, Switzerland). The sample with different water contents was collected and put
into an electro-thermostatic blast oven at 60 °C for dried, then analyzed by developed
quantitative models.
2. 2. 4. 2 Phase transition in wet granulation of FXD alone
To assess the influence of the amount of water used for granulation on phase
transition behavior of pure FXD, samples of FXD with different water contents (0%,
5%, 10%, 15%, and 20%) were prepared by wet granulation. About 150 g of pure FXD
was weighed and granulated in 2 L granulator at impeller speed of 150 rpm and chopper
speed of 500 rpm, then added 26.08 g of purified water to the powder by spray method
for preparing the sample with 5% water content. Then different amount of water was
added in the same batch with the same manner for preparing samples with 10%, 15%,
and 20% water content. After each process of adding water, the granulation times were
set as 1 min, and then about 3 g of the sample with different water contents was taken
out. Then 30 mg of the sample was accurately weighted, whose water content was
measured by Karl Fischer Titration. The remaining sample was put into an electro-
thermostatic blast oven at 60 °C for fully dried, which were then collected for further
analysis by the validated NIR and DSC analysis techniques. The actual amount of added
water and the corresponding water content of wet mass were listed in Table S1. The
water content of the sample by wet-granulation process was selected as variable in
quantitative analysis rather than the amount of added water because of the slight loss
during the water addition process by spraying system.

9
2. 2. 4. 3 Phase transition in wet granulation of formulation
To assess the phase transition of drugs in formulation, 200 g of the formulation
powder was pre-mixed in a granulator equipped with a bowl of 2 L at stirring paddle
and cutter speeds of 100 rpm and 250 rpm, respectively. The formulation composed of
the FXD (40%, w/w), pregelatinized starch (40%, w/w), microcrystalline cellulose
(17%, w/w), and croscarmellose sodium (3%, w/w). Samples of formulation with
different water contents (10%, 15%, 20%, 25%, 30%, 35% and 40%) were prepared by
wet granulation in similar fashion as described in 2.2.4.2 section, dried and further
analyzed by the validated NIR and DSC analysis techniques. The actual amount of
added water and the corresponding water content of samples in the formulation were
listed in Table S1.
2. 2. 4. 4 Phase transition in wet granulation of FXD-excipient mixtures
In order to determine the possible influence of excipient in the conversion of FXD,
simplified physical mixtures containing the FXD and single excipient (pregelatinized
starch /and microcrystalline cellulose) were prepared, presented 1:1 of mass fraction
range with total weight of 200 g. The operating conditions, containing bowl volume,
impeller speed and chopper speed were the same as the formulation mentioned in
section 2.2.4.3. FXD-excipient mixtures with different water contents were prepared
using the same steps as in 2.2.4.2 section, and analyzed by the validated NIR
spectroscopic techniques. The actual amount of added water and the corresponding
water content of FXD-excipient mixtures were listed in Table S1.
3. Results and discussion
3. 1 Solid state characterization of polymorphs
As observed in PXRD patterns Figure 3A, the characteristic peaks of Form I and
Form II are obviously different and the results are consistent with those reported in the
literature [26, 31]. The primary difference of FT-IR spectra between Form I and Form
II of FXD (Figure 3B) was observed for OH symmetrical stretching at 3400 cm−1 and
several shifts in the fingerprint region between 400 cm−1 and 1800 cm−1. These
observations well corresponded with those reported in the literature [27].
In DSC thermogram Figure 4A, FXD Form I showed a sharp melting peak at 195.9
°C (onset T) (endotherm peak 202.6 °C), which was in good agreement with the
reported melting point values of hydrochloride salt FXD Form I in the range of 193 °C
– 199 °C [26]. While hydrates, FXD Form II showed a dehydration peak at about 100 °C,

10
a small endothermic peak at 132 °C and no melting peak at 195.9 °C, even no
disturbance of excipients melting events at the point of 195.9 °C was observed. Hence,
this significant difference in thermal behavior of both forms makes DSC thermal
analysis useful for quantitative analysis of polymorphic conversion.
From the NIR spectra of the FXD Form I and Form II (Figure 4B), we can see an
intense difference at 5150 cm−1 which is related to the water absorption bands (OH
stretching coupled with angular deformation of OH) [27]. The spectra of Form I could
be discriminated from Form II. The difference in the spectra makes NIR suitable for
qualitative and quantitative analysis of FXD polymorphs.
3. 2 Dynamic vapor sorption (DVS) of polymorphs
Hygroscopicity is a crucial property in pharmaceutical development as it has a
direct impact on other properties such as solubility, dissolution rate, and stability [32].
Figure 5 shows DVS isotherm plots of FXD Form I and II and formulation powder
containing Form I or Form II at 25 °C. Since the mass change of original FXD (Form
I) was under 0.4% at RH 90%, it indicated that Form I had no hygroscopicity property.
However, FXD Form II has a weight gain of 7.2% at RH 20%, reaching 11.8% at RH
90%. The moisture uptake by Form II is more than 200 times higher than that of Form
I at RH 20%. In addition, DVS analysis of formulation containing Form I or Form II
shows that moisture uptake by Form II is twice higher compared to that of Form I at
RH 20%. The results suggested that phase transition of FXD affected sample properties
of powder surface and water absorption dynamics. DVS results showed that conversion
to Form II might alter stability of the pharmaceutical product.
3. 3 Quantification of FXD Form I
3. 3. 1 NIR model calibration and validation
NIR spectroscopy has been widely used in the qualitative and quantitative analysis
of polymorph mixture. The method, wavelength and pretreatments can make automatic
select and calibrate using NIRCal calibration software guide (Calibration Wizard).
Followed the guide, spectra were collected to develop the model for quantification of
FXD Form I in binary mixtures and multicomponent mixtures. The main quality
parameters used to evaluate the model were Q value, R2, RMSEC, RMSEP and SEE.
Several chemometrics techniques are applied for quantification in spectra processing,
such as MLR, PCR, and PLS regression approaches. Based on Q value, partial least
squares regression (PLS) was selected spectra processing for quantification of FXD

11
Form I. The original and pretreatment spectra are presented in the Figure S2. The
optimal model parameters are shown in Table 1. The R2 value of 0.9994 and 0.9988
were obtained from PLS regression for binary mixtures and multicomponent mixtures,
respectively. The significant difference between SEC and SEP indicate the inadequacy
of the model [28]. And in our study, the difference between SEC and SEP was less than
0.1 for developed models. Additionally, the consistency was defined as the ratio
between the SEC and the SEP. This parameter should be around 0.8-1.2 in NIR of Buchi,
indicating an optimal balance between the C-set and the V-set [33]. The RMSEC and
RMSEP values of 0.59 and 1.13 for binary, 1.14 and 1.04 for multicomponent mixtures
were obtained. The model is considered to provide good fitting results when the
parameters of calibration and prediction are close to each other. Figure 6A - B shows
the plot of predicted versus the reference content of FXD Form I. The test points were
distributed close to the straight line (slope = 1 and intercept = 0), with little deviation,
indicating a good linear relationship between the predicted and reference values.
The validation samples containing 10%, 50% and 80% FXD Form I were used to
evaluate the accuracy between the predicted content of Form I by NIR model and
reference value. Validation data of FXD Form I in binary mixtures and multicomponent
mixtures by NIR calibration model were shown in Table 3. The predicted amount of
FXD Form I was close to the actual amount. Additionally, the accuracy of model was
evaluated by recovery%. The accuracy of the model in predicting the content of Form
I is highly dependent of the choice the drug content, especially for determining the low
level of FXD Form I. Precision of the proposed method was determined by measuring
the repeatability of 50% w/w Form I sample. The % RSD values were 1.8 for binary
and 1.5 for multicomponent mixtures. As a conclusion to the validation of the proposed
method, the development was able to determine the content of Form I present in binary
and multicomponent mixtures with adequate accuracy and precision.
3. 3. 2 DSC model calibration and validation
0%, 10%, 30%, 50%, 60%, 80%, and 100% FXD Form I in binary mixtures and
0%, 20%, 40%, 60%, 80%, 90%, and 100% FXD Form I in multicomponent mixtures
(formulation) were tested by DSC. The DSC curves were recorded in Figure S3. As
shown in Table 2 for binary mixtures system, the linear regression equations and values
of coefficients of determination R2 were y = 0.8979 x – 0.9663; R2 = 0.9985. And for
multicomponent mixtures system, the regression equation was y = 0.8003 x + 1.2157;

12
R2 = 0.9957. The results showed that the FXD Form I content % and enthalpy had a
positive correlation and had a good linear relationship. The plot of calculated values
versus reference content of FXD Form I % is depicted in Figure 6 C-D.
The accuracy (recovery%) of DSC linear equations method was evaluated by
validation samples containing 10%, 50%, and 80% Form I. The actual Form I value and
the calculated Form I by DSC linear model were shown in Table 3. The recovery (%)
was low at low concentration of FXD Form I, mainly due to small sample content. The
% RSD values (precision) were 2.4 for binary and 3.6 for multicomponent mixtures.
These results suggested that a good calibration DSC model based on the melting point
was achieved.
3. 3. 3 Quantification of Form I in excipient-FXD mixtures by NIR method
It has been demonstrated that excipient can significantly affect phase transition in
formulation during wet granulation. They can hinder unwanted phase transition and
ensure the required stability of the drug in the formulation [34]. The results obtained
from chemometrics techniques were presented in Table 1. The raw and pretreated
spectra can be seen in supplementary Figure S4. And Figure 6 E-F show the NIR
calibration model between reference values and predicted values obtained by
chemometrics techniques. All samples are distributed around the target line
demonstrating agreement between the predicted and nominal values. All those results
demonstrated that NIR method was suitable for quantitation of FXD Form I in
excipient-FXD mixtures.
The accuracy and precision by NIR calibration model for excipient-FXD mixtures
were shown in Table 4. Similarly, the accuracy is low at low concentrations of FXD
Form I in excipient-FXD mixtures. The flowability of FXD was very poor, leading to
the possibility of uneven distribution at low FXD Form I content (< 10%). The %RSD
value was observed to be 3.8 for pregelatinized starch-FXD polymorphs forms mixtures
(50% w/w) and 3.6 for microcrystalline cellulose-FXD polymorphs forms mixtures
which confirmed that NIR method was precise. Quantification of Form I in excipient-
FXD mixtures can be rapidly achieved using NIR chemometric models.
3. 4 Application of Quantification method
The developed and validated NIR and DSC methods were employed to identify
and quantify the degree of phase transition of FXD during wet granulation. In addition,
it was worth mentioning the choice of the drying temperature. DSC result of Form II

13
indicated that the hydrate form lost water and converted to the anhydrous form at about
120 °C. Moreover, the stability of Form II at high temperature (60 °C) for 30 days was
investigate via PXRD (supplied as supporting information in Figure S5), which
demonstrated that Form II was stable without secondary phase transition at 60 °C.
Consequently, it was suitable to set the drying conditions at 60 °C.
3.4.1 Solid-state phase transition in wet granulation of pure FXD
Figure 7A shows the FXD Form I content were predicted by NIR and DSC
quantitative analysis methods during pure FXD wet granulation process. It was
observed %FXD Form I predicted by two analysis techniques were similar and
decreased with the water content. The transformation started within 5% water content,
reaching 50% at about 6% water content, showing a significant transformation rate.
And complete transformation to Form II was observed at about 15% water content.
When pure FXD was involved in wet granulation, the phase transition process was very
intense.
3. 4. 2 Solid-state phase transition in wet granulation of formulation
The developed and validated NIR and DSC methods were used to quantify the
Form I content during formulation wet granulation process. It was observed the
variation trend of % FXD Form I predicted by two analysis techniques with water
content was similar. Figure 7B shows that no significant change of Form I content was
observed when the water content varies from 0% to 15%. At above 25% water content,
percentage of Form I was reduced to 50%. The complete phase transition was observed
at 40% water content. The phase transformation of FXD in formulation was retarded
due to the presence of excipients in comparison to the results presented in Figure 7A.
Studies have shown that wet granulation of FXD induces phase transition, which
depends not only on the amount of water used for granulation, but also on the selection
of appropriate excipients for granulation. In addition, research on the variation trend of
% FXD Form I with water content is meaningful for the development of FXD generic
drugs. If the degree of phase transition in the reference preparation is determined, the
proper process parameter - the amount of water added was guided in the granulation
process. In other words, the relationship between the phase transition degree of FXD
and water content can be a tool to determine the optimum quantity of water in wet
granulation.
For application, both NIR and DSC have successfully quantified the phase

14
transition of FXD in pure API and formulation during wet granulation. Although NIR
(spectroscopic technique) and DSC (thermodynamic analysis) belong to different
quantification system, the variation trend of FXD Form I predicted by two analysis
techniques with water content was similar. For detecting the low level of FXD Form I,
the accuracy of both quantification techniques was impacted. Based on the
consideration of detection efficiency and predictability, NIR may be the best choice for
quantification of FXD polymorphs. Our current study has determined that the phase
transition of FXD affect the quality attributes of granules and the quality of final
products (data unpublished). The quantification of FXD phase transition is critical. The
phase transition needs to be controlled within a certain range.
3. 4. 3 The effect of excipient on phase transition of FXD in wet granulation
In this part of this study, the aim is to investigate the effect of excipient in wet
granulation process with different water content for the phase transition of FXD. To
anticipate and prevent such product inconsistencies, it is necessary to understand the
phase transition mechanisms of API and excipients. In Figure 8, the percentage FXD
Form I predicted by calibration NIR models is plotted as a function of water content.
As given in Figure 8, no significant Form I conversion occurs at below 15% water
content in FXD wet granulation containing pregelatinized starch (1:1), reaching 50%
transformation at about 20% water content, and complete transformation was observed
at about 30% water content. Microcrystalline cellulose was able to hinder the phase
transition of FXD at low water content (< 10%), not at the amount of water needed to
form granules. At 13% water content, the FXD Form I% decreased rapidly to 70% in
the microcrystalline cellulose formulation, reaching 50% transformation at about 15%
water content. The complete phase transition was observed at about 25% water content
in FXD wet granulation containing microcrystalline cellulose, showing a accelerate of
conversion process, in comparison to FXD wet granulation containing pregelatinized
starch. Microcrystalline cellulose was partially crystalline excipient. Water molecules
penetrate cellulose, particularly in the amorphous regions. Once microcrystalline
cellulose has absorbed water to a certain point, further addition of water will be resisted
and present on the surface of particles [35]. This may be the reason why the phase
transition of FXD intensifies in the range of 10% to 20% water content in the
microcrystalline cellulose formulation. Pregelatinized starch was corn starch with
amylopectin (approximately 73%, soluble, gelatinized) and amylose (approximately

15
27%, insoluble). Pregelatinized starch have a strong affinity than microcrystalline
cellulose for water molecules [34, 36]. The dynamic vapor sorption isotherm of
pregelatinized starch and microcrystalline cellulose were presented in Figure S6.
Therefore, stability of FXD increased in the formulation containing pregelatinized
starch than microcrystalline cellulose.
The excipients in the formulation hindered hydrate formation of FXD at low water
contents. Results showed that the availability of using less-crystalline excipient in
formulation to manipulate phase transformation of active pharmaceutical ingredient
which was sensitive to water used in wet-granulation. Thus, excipients might
significantly affect solid-state phase transformation and participate actively in
improving the characteristics of formulations during wet granulation [37]. In our study,
the effects of single excipient on water-mediated phase transformation of FXD was
quantified successfully by NIR, which could provide a reference for screening the
proper excipient, filtrating the kinds and ratio of the excipients and optimizing the
formulation and preparation. Based on the method, if we can have a good acknowledge
of the risks involved in manufacturing and how to mitigate those risks, it will be
beneficial to the development and design of products.
4. Conclusions
It is important to understand the product and process along a knowledge of the
risks involved in manufacturing, particularly when crystal forms change during
production. In this study, we successfully identified and quantified the FXD Form I in
pure API and physical mixtures formulation during wet granulation by using NIR and
DSC quantitative analysis methods. A relationship between the degree of FXD phase
transition and water content was established in wet-granulation process. The
relationship can be used as a tool to optimize the water amount added and guarantee
the consistency of final product quality. Moreover, the knowledge gained from this
study provided a reference value of feasibility for online real time study by NIR or
Raman in production. In addition, we observed transformation of FXD to hydrate Form
II in the formulation in response to the addition of water occurred at a slow rate than
pure FXD. This result indicated the presence of excipients hinder phase transition of
FXD in wet granulation. The effect of excipient on phase transition of FXD was
quantified by NIR. The stability of FXD increased in the formulation containing
pregelatinized starch than microcrystalline cellulose. This study demonstrated that it

16
was important to understand the effect of the water amount added and excipient
selection on solid-state phase transition in formulation during wet granulation.

Conflicts of interest
The authors declare no conflicts of interest
Acknowledgement
We thank the Research and Development group of Liaocheng High-Tech
Biotechnology Co., Ltd. for excellent technical assistance. This work is financially
supported by the National Science and Technology Major Project (no. 2017ZX09201-
003), the Open Project of Shandong Collaborative Innovation Center for Antibody
Drugs (No. CIC-AD1828) and Tai-Shan Scholar Research Fund of Shandong Province
of China. The above work was also technically supported by Shandong Province
Collaborative Innovation Center for Antibody Drugs, Shandong Province Engineering
Research Center for Nanomedicine and Drug Delivery Systems and Shandong Province
Engineering Laboratory of Anti-Viral Drugs.
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Highlights

20
• Quantification of polymorphic forms of FXD by NIR and DSC technologies.
• Analytical performance of quantitative methods was evaluated.
• Quantitative methods were used to monitor phase transition during wet granulation.

Figure 1. Chemical structure of Fexofenadine Hydrochloride.

Figure 2. Example of a DSC curve evaluation. The enthalpy of drug was determined using the
sigmoidal baseline type.

21
 A Form II Form I Form I Form II
B
5.9 7.4 14.2 18.2

transmittance (%)
Intensity

5 10 15 20 25 30 35 40 4000 3500 3000 2500 2000 1500 1000 500

2 theta Wavenumber (cm-1)

Figure 3. PXRD and FT-IR overlay of the FXD solid forms.

A Form I Form II B Form I Form II

0.0

-0.5
Heat Flow (W/g)

Absorbance

-1.0

-1.5

-2.0

-2.5
20 40 60 80 100 120 140 160 180 200 220 10000 9000 8000 7000 6000 5000 4000
Temperature T (°C) Wavenumber (cm-1)

Figure 4. Thermogram and spectra of FXD Form I and Form II obtained from DSC (A) and NIR (B).
20

FXD Form I
FXD Form II
15 FXD Form I formulation
FXD Form II formulation
Change in Mass (%)

10

0 10 20 30 40 50 60 70 80 90 100
Target % P/P0

Figure 5. Dynamic vapor sorption isotherm of FXD Form I and II, excipients and formulation powder
22
 containing Form I or Form II at 25 °C.

NIR Spectroscopy for binary and multicomponent mixtures

100 100
A binary of FXD polymorphs forms B multicomponent mixtures of FXD formulation
80 80
Predicted FXD Form I (% w/w)

Predicted FXD Form I (% w/w)

60 60

40 40

20 20

0 0

0 20 40 60 80 100 0 20 40 60 80 100
Reference FXD Form I (% w/w) Reference FXD Form I (% w/w)

DSC analysis for binary and multicomponent mixtures

100 100
C binary of FXD polymorphs forms D multicomponent mixtures of FXD formulation
80 80

Predicted FXD Form I (% w/w)

Predicted FXD Form I (% w/w)

60 60

40 40

20 20

0 0

0 20 40 60 80 100 0 20 40 60 80 100
Reference FXD Form I (% w/w) Reference FXD Form I (% w/w)

NIR Spectroscopy for excipient-FXD mixtures

100 100
E pregelatinized starch-FXD polymorphs forms mixtures F microcrystalline cellulose-FXD polymorphs forms mixtures

80 80
Predicted FXD Form I (% w/w)
Predicted FXD Form I (% w/w)

60 60

40 40

20 20

0 0

0 20 40 60 80 100 0 20 40 60 80 100
Reference FXD Form I (% w/w) Reference FXD Form I (% w/w)

Figure 6. Plots of reference values versus predicted values obtained by the NIR chemometrics
techniques and DSC analytical techniques.

23
 A B
100 100

80 NIR 80
DSC NIR
DSC
% FXD Form I

% FXD Form I
60 60

40 40

20 20

0 0
0 5 10 15 20 0 5 10 15 20 25 30 35 40 45
water content (%) water content (%)

Figure 7. Percentage FXD Form I predicted by NIR and DSC Quantification methods after wet
granulation (A) pure FXD and (B) formulation. The line is drawn to assist in visualizing the trend.

100
FXD + pregelatinized starch
FXD + microcrystalline cellulose
80
% FXD Form I

60

40

20

0
0 5 10 15 20 25 30 35
water content (%)

Figure 8. Percentage FXD Form I predicted by calibration NIR models after wet granulation. The line
is drawn to assist in visualizing the trend.

Declaration of interests

The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:

24
 Credit Author Statement

Suye Li: Conceptualization, Methodology, Data curation, Validation, Investigation,

Writing- Original draft preparation

Yanna Zhao: Methodology, Writing- Reviewing and Editing

Lili Wang: Methodology, Validation, Investigation

Hengqian Wu: Methodology, Validation, Investigation
Yan Gao: Investigation
Lingxuan Zhang: Investigation
Zhengping Wang: Writing- Reviewing and Editing

Jun Han: Writing- Reviewing and Editing, Supervision

25