Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305

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Journal of Pharmaceutical and Biomedical Analysis

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

Pentabromobenzyl-RP versus triazole-HILIC columns for separation of

the polar basic analytes famotidine and famotidone: LC method
development combined with in silico tools to follow the potential
consequences of famotidine gastric instability
Rania El-Shaheny a,b,∗ , Mohamed O. Radwan c,d,e,∗∗ , Fathalla Belal a , Koji Yamada f
a
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt
b
Department of Hygienic Chemistry, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki, 852-8521, Japan
c
Medicinal and Biological Chemistry Science Farm Joint Research Laboratory, Faculty of Life Sciences, Kumamoto University, 5e1 Oehonmachi, Chuo-ku,
Kumamoto, 862-0973, Japan
d
Department of Drug Discovery, Science Farm Ltd., 1-7-30 Kuhonji, Chuo-ku, Kumamoto, 862-0976, Japan
e
Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Research Division, National Research Centre, Dokki, 12622, Cairo,
Egypt
f
Medicinal Plant Laboratory, Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki, 852-8521, Japan

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

Article history: The competence of hydrophilic interaction (HILIC) and reversed phase liquid chromatography (RPLC)
Received 2 December 2019 modes, employing two new stationary phases: triazole- and pentabromobenzyl-bonded silica (PBr),
Received in revised form 7 March 2020 respectively, was inspected for separation of two polar basic analytes: famotidine (FAM) and its acidic
Accepted 7 April 2020
degradant famotidone (FON). Comparison of the chromatographic efficiency, greenness, and economy
Available online 18 April 2020
aspects showed that the RPLC is superior to the HILIC. Hence, the RPLC method was adopted and validated
adhering to the FDA guidelines showing excellent linearity for FAM (1.0−20.0 ␮g/mL) with a detection
Keywords:
limit of 0.14 ␮g/mL. The method was applied to study the behavior of FAM in simulated gastric juice
Famotidine
Famotidone
(SGJ), where it exhibited rapid degradation yielding FON. This degradation pathway is a probable major
RPLC reason for the poor bioavailability of FAM. The kinetic study of the gastric degradation of FAM in SGJ
HILIC demonstrated pseudo-first order reaction with a rate constant of 8.1 × 10−3 min-1 . Moreover, FAM degra-
Gastric instability dation has been proven to be pH-dependent and catalyzed by the gastric juice components. Hence, in situ
In silico tools buffered dosage form is recommended to overcome or decrease this problem. Molecular docking study
shows that FON is missing a crucial stabilizing interaction with the key amino acid Asp98 causing a
reduced activity at hH2 R receptor relative to FAM. Moreover, ADMET properties prediction revealed
some differences in the toxicity, pharmacokinetics, metabolism, and solubility profiles of FAM and FON.
© 2020 Elsevier B.V. All rights reserved.

1. Introduction RP columns while highly polar or ionic molecules with high

Reversed-phase liquid chromatography (RPLC) is a key tech-
nique for the separation and analysis of a vast range of compounds.
However, it is not applicable for all types of analytes. Compounds
with adequate lipophilicity could be retained on the common
∗ Corresponding author at: Department of Pharmaceutical Analytical Chemistry,
Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt.
∗∗ Corresponding author at: Medicinal and Biological Chemistry Science Farm Joint
Research Laboratory, Faculty of Life Sciences, Kumamoto University, 5e1 Oehon-
machi, Chuo-ku, Kumamoto, 862-0973, Japan.
E-mail addresses: rania yomna@mans.edu.eg, rania n2010@yahoo.com
(R. El-Shaheny), mohamedradwan@kumamoto-u.ac.jp (M.O. Radwan).
hydrophilicity are not adequately retained and eluted nearby the
column void volume [1]. The analysis of ionized bases that con-
stitutes greater than 70 % of pharmaceuticals, by RPLC suffers also
from many difficulties, for example interaction with the stationary
phase silanol groups and overloading which both cause poor peak
shapes [2].
Some approaches have been adopted to enhance the retention of
polar and/or basic compounds on RP columns such as derivatization
or using ion-pair chromatography (IPC). Another chromatographic
approach to improve the retention of such challenging analytes is
ion-exchange chromatography (IEC). Yet, these approaches entail
some drawbacks. For example, the derivatization techniques need
multiple steps and lengthy time [1], while IPC and IEC are applica-

https://doi.org/10.1016/j.jpba.2020.113305
0731-7085/© 2020 Elsevier B.V. All rights reserved.
2 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305
ble only for ionizable compounds and the complex nature of their
mobile phases leads to irreproducibility compared to simpler ones.
Moreover, these two chromatographic modes are not compatible
with mass spectrometric detection because of the ion-suppression
in the electrospray ionization in case of IPC and the contamination
of the spectrometer with the high salt concentration in the mobile
phase in case of IEC [1,3,4].
Modern developments in LC stationary phases provide some
superior alternatives for the separation of polar and basic com-
pounds. For this purpose, some polar stationary phases that are
compatible with RPLC have been developed. Among these sta-
tionary phases is the newly emerged pentabromobenzyl-bonded
silica (PBr) which enables the separation by London dispersion
force interactions that contribute to intermolecular force among
all molecules whether polar or non-polar [5]. Thus, compounds
with little hydrophobicity, which are not retained on traditional RP
columns, could be separated by dispersion force interactions using
the PBr column.
Another evolving approach for separation of polar and basic ana-
lytes is the hydrophilic interaction liquid chromatography (HILIC)
that uses a polar stationary phase and a mobile phase containing
a mixture of aqueous buffer (≥5%) and a less polar solvent (mostly
acetonitrile, ACN ≥ 60 %). The mechanism of HILIC separation is
complex including partitioning, ion exchange (IE), and hydropho-
bic retention [6]. HILIC provides several advantages for the analysis
of polar compounds such as selectivity, good retention, reasonable
peak shape, and possibility of using high flow rates due to high
organic solvent content in the mobile phase. In this connection, a
new HILIC column packed with a triazole-bonded silica has been
recently manufactured and assigned for the separation of polar
and highly polar compounds of different natures (acidic, basic, neu-
tral, and amphoteric). The main interaction force involved in this
stationary phase is the hydrophilic interaction with a weak anion
exchange ability [7].
The aim of the present study is to compare the performance of
HILIC and RPLC modes employing the new triazole- and PBr-bonded
stationary phases, respectively, for the separation of selected
polar basic analytes to explore the most fitted method in terms
of column efficiency, selectivity, analysis time, costs, and eco-
harmony. Famotidine, 3-[[[2-[(diaminomethylene)amino]thiazol-
4-yl]methyl]sulfanyl]-N-sulfamoylpropanimidamide [8] (FAM) is
the candidate of this study since it is a highly polar basic com-
pound with a partition coefficient (log PO/W ) of -0.64 and a pKa
of 7.06 [9]. It is a histamine H2 -antagonist that is widely used
as a therapy for benign gastric and duodenal ulceration, gastro-
esophageal reflux disease and Zollinger-Ellison syndrome [10].
FAM is highly susceptible to acidic degradation [11,12] yielding
3-[[[2-[(diaminomethylene)amino]thiazol-4- yl]methyl]sulfanyl]-
N-sulfamoylpropanamide [8], also known as famotidone (FON),
that is identified by the United States Pharmacopoeia (USP) [13] and
the British Pharmacopoeia (BP) [8] as a specified impurity in FAM
pure powder and pharmaceuticals. To the current date, most of the
reported HPLC methods for FAM [14–18], including the Compen-
dial methods [8,13], depended on IPC or employed aqueous-rich
mobile phases which cause de-wetting of RP columns leading to
poor retention, low selectivity, and reproducibility problems [1].
These concerns motivated the authors to conduct a comparative
chromatographic study for inspection of the retention character-
istics of FAM and its related compound, FON, on the two cited
stationary phases, triazole- and PBr-bonded silica, to test their sep-
aration power for discrimination of these two closely-related polar
basic compounds.
According to the results of this study, the more efficient chro-
matographic mode was applied to investigate the gastric stability
of FAM and to shed light on the kinetics of its degradation to FON
in simulated gastric juice (SGJ). This investigation is very impor-
tant since FAM exhibits a poor bioavailability of about 20 to 66%
[9], thus it is mandatory to explore whether the drug degrada-
tion in gastric juice has a contribution to the poor bioavailability or
the major cause is the poor intestinal permeability only [19]. The
ADMET (absorption, distribution, metabolism, excretion, and toxi-
city) properties and receptor binding affinity of FON have been also
investigated by in silico tools and compared to FAM itself for better
understanding of the potential results of gastric-induced degrada-
tion of FAM and its fate in human body.
2. Experimental
2.1. Equipment
LC separation was performed with a Hitachi HPLC setup (Tokyo,
Japan) consisted of 655A-11 liquid chromatograph, a high sensi-
tivity series L-4000H UV-detector, D-2500 chromato-integrator,
LC-organizer, and a Rheodyne injector valve with a 50-␮L sample
loop. An online Gastorr BG-34 solvent degasser (FLOM Corporation,
Tokyo) was integrated into the system. The pH measurement was
done using SK-620 pH-meter (Sato Keiryoki MFG Co. Ltd, China). A
BT-15 Yamato thermostatically-controlled water bath (Tokyo) and
As One ultrasonic bath (Osaka, Japan) were used.
2.2. Software
The amino acid sequence of human H2 receptor (hH2 R) P25021
was retrieved from the Universal Protein Resource (Uniprot)
(https://www.uniprot.org/). The crystal structure of turkey ␤1 -
adrenoceptor was obtained from Protein Data Bank (PDB entry:
2vt4) [20] and used as a model. The sequence alignment, homology
model, and geometry validation of amino acids, and docking pro-
cess of FAM and FON into the generated model were performed by
Molecular Operating Environment MOE 2018.01 (Chemical Com-
puting Group, Montreal, Canada) utilizing its default parameters
[21]. ADMET PredictorTM 9.0 from Simulation Plus, Inc. (CA, USA)
was used to predict The ADMET properties. HPLC-Environment
Assessment Tool (HPLC-EAT) was applied using the software
developed by Gaber and co-workers [22]. Statistical analysis was
accomplished with Microsoft Office Excel 2013.
2.3. Materials and reagents
Famotidine pure sample (Lot No. FXLHF-HC) with a certified
purity > 98.0 % was purchased from Tokyo Chemical Industry Co.,
Ltd. (Tokyo). Ammonium acetate, sodium chloride, and pepsin from
porcine gastric mucosa (powder ≥250 units/mg solid) were pur-
chased from Sigma-Aldrich (St. Louis, MO, USA). Acetonitrile (ACN)
(HPLC grade) was obtained from Kanto Chemical Co., Inc. (Tokyo).
Concentrated HCl (35–37 % w/v) and glacial acetic acid were pur-
chased from Nacalai Tesque (Kyoto, Japan). Preparation of SGJ (pH
1.2) was done as per the USP [13] by dissolution of 2 g NaCl and
3.2 g of pepsin in 7 mL HCl and making up the final volume to 1.0 L.
Solution A containing 0.2 % NaCl and 0.32 % pepsin in 0.06 % acetic
acid (pH 3.5) was also prepared to be used as a substitute of SGJ in
the validation study to avoid degradation of FAM.
2.4. Chromatographic conditions
Cosmosil® HILIC packed column (250 mm × 4.6 mm ID, 5 ␮m
particle size) and Cosmosil® PBr packed column (150 mm × 4.6 mm
ID, 5 ␮m particle size), from Nacalai Tesque Co., were used in the
comparative chromatographic study. Mobile phases used in this
study were prepared by mixing ACN and ammonium acetate buffer
in various proportions. Acetic acid was used for pH adjustment.
Mobile phases were degassed via ultra-sonication for 30 min and
 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305
filtered with 0.45 ␮m membrane filter. UV-detection was carried
out at 267 nm for simultaneous sensitive detection of FAM and FON
[8]. The optimum mobile phase finally selected for analytical appli-
cations was ACN: 0.01 M ammonium acetate buffer (25:75, v/v), pH
6.3 at a flow rate of 1 mL/min using the PBr column.
2.5. Stock solution and resolution solution
For the calibration and validation study, FAM stock solution
(100.0 ␮g/mL) was prepared in Solution A. This solution was stable
up to 5 days without any degradation when kept in a refrigerator
at 4 ◦ C. During the separation study, a resolution solution was pre-
pared so as to contain a mixture of FAM and FON, by dissolving
FAM (10.0 mg) in 50 mL of 0.06 M HCl, this solution was incubated
for 30 min at 37 ◦ C in a thermostatically-controlled water bath. A
volume of 0.75 mL of this solution was transferred to 10-mL volu-
metric flask, neutralized with 0.03 M Na2 CO3 , and made up to the
final volume with the respective mobile phase for the HILIC or the
RPLC method. This solution was found stable without further degra-
dation of FAM for 5 days when kept in the refrigerator at 4 ◦ C. All
flasks were wrapped with aluminum foil for protection from light
to avoid incorporation of any additional degradation factor.
2.6. Calibration curve
Calibration curve was constructed using 100.0 ␮g/mL solution
of FAM prepared in Solution A as a substitute for SGJ due to the
high instability of FAM in it. Increasing aliquots of FAM solution
(10.0–200.0 ␮L) were transferred to a series of glass vials and made
up to 1 mL with the mobile phase to obtain final concentrations
within the range of 1.0−20.0 ␮g/mL. Solutions were mixed well,
filtered through 0.45 ␮m membrane syringe filter, and 50 ␮L were
injected in triplicate. The calibration curve was constructed by plot-
ting the average peak areas versus drug concentration (␮g/mL) and
the linear regression equation was calculated.
Fig. 1. Illustration of the mechanism of interaction and separation of FAM and FON on (A) HILIC and (B) PBr stationary phases.
3
2.7. Procedures for studying the stability of FAM in simulated
gastric juice
Twenty mg of FAM pure powder was accurately weighed,
dissolved in 50 mL of SGJ (pH 1.2), and incubated in a
thermostatically-controlled water bath at 37 ◦ C. Aliquots of 25 ␮L
of the incubation mixture were taken at specific time-intervals
(10−160 min), neutralized with 25 ␮L of 0.03 M Na2 CO3 , and com-
pleted to 1 mL with the mobile phase. Solutions were mixed well,
and filtered through 0.45 ␮m membrane syringe filter. Fifty ␮L
were injected (triplicate) and eluted under the optimum chromato-
graphic conditions.
For comparing the stability of FAM in SGJ with variable pH val-
ues, a modification of the USP method [13] was done to prepare
SGJ of pHs 1.6 and 2.0 using 0.01 and 0.005 M HCl, respectively.
Moreover, the experiment was also carried out in 0.06 M HCl for
comparison with SGJ (pH 1.2). Then, the same procedure described
earlier was performed using these solutions. A control experiment
was also conducted using Solution A (SGJ of pH 3.5) where no degra-
dation of FAM was evident.
The observed pseudo-first order rates of FAM degradation to
a
FON (k) were calculated from the semi-logarithmic plots of log a−x
versus time (t) according to Eq. (1) [23]:
a
kt = 2.303 log (1)
a−x
Where (a) is the initial drug concentration, (a–x) is the remaining
drug concentration, (t) is the time (min), and (k) is the degradation
rate constant (min−1 ).
Also, the half-life time (t1/2 ) was calculated according to Eq. (2)
[23]:
0.693
t1/2 = (2)
k
4 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305
3. Results and discussion
In this study, HILIC and RPLC modes, employing newly com-
mercialized stationary phases were compared for separation of
two polar basic compounds; FAM and FON (Fig. 1). The reported
log P(O/W ) and pKa values of FAM are -0.64 and 7.06 [9], respec-
tively, while those of FON were calculated by the ADMET predictor’s
physicochemical and biopharmaceutical (PCB) module [24] to be -
1.172 and 6.86, respectively. Thus, it seems a challenge to achieve
good separation and satisfactory chromatographic retention of
these two related compounds by conventional RPLC method. In
next sections, we discuss the separation of the two compounds by
HILIC and RPLC utilizing triazole- and PBr-bonded silica columns,
respectively.
3.1. HILIC separation of FAM and FON on triazole-bonded silica
column
The separation of FAM and its acidic degradation product, FON,
by the HILIC technique was studied using the triazole-bonded silica
column (HILIC). This stationary phase is assigned for the separa-
tion of basic, acidic, or neutral compounds [7]. Generally, HILIC is
a multi-mechanistic separation mode based mainly on hydrophilic
partitioning of solutes between the organic-rich mobile phase and
the static aqueous layer on the stationary phase surface. The more
polar analyte will associate to a greater extent to this layer and
eluted later and the reverse for the less polar analyte. Secondary,
the HILIC separation is also affected by electrostatic interaction
between the charged analyte and stationary phase. Hence, the
mobile phase pH has a significant contribution to the separation
process by controlling the ionization of both the analytes and the
stationary phase.
In this study, the separation was investigated using the reso-
lution solution described in “Section 2.5”. First, the effect of pH of
ammonium acetate buffer on the separation and performance char-
acteristics was investigated. Due to the apparent change of buffer
pH up on mixing with ACN, the pH was measured after mixing
(Table S1, Supplementary material). As illustrated in Fig. 2A, the
change in pH has a limited effect on the retention of FAM, while hav-
ing a profound effect on the retention of FON. Though, the degree
of ionization of FAM, as a basic drug, is dependent on the pH of the
mobile phase according to the following equation [23]:
100
% Ionization =
[1 + antilog (pH − pka)]
Despite the variable degrees of ionization of FAM to cationic
species with the change in the mobile phase pH: 82.03 % ionization
at pH 6.4 versus 22.39 % ionization at pH 7.6 (Supplementary mate-
rial, Table S1), its retention was almost unaffected. Considering the
chemistry of the HILIC column could explain this behavior since
it is packed with 1,2,4-triazole-modified silica (Fig. 1A) that can be
positively charged over the whole studied pH range where its pKa is
10.26 [25]. As a result, the stationary phase would interact strongly
with the negatively charged analytes by electrostatic interaction,
while no attraction occurs with positively charged species such as
FAM [1]. Meanwhile, the changeable retention of FON with varia-
tion in the pH is possibly attributable to the variable electrostatic
interaction of the electronegative carbonyl oxygen of FON with the
positively charged stationary phase, where decreasing the pH of
the mobile phase resulted in association of the carbonyl oxygen to
a proton (Eq. (4)) [26] leading to a weaker interaction with the HILIC
stationary phase and a decreased retention of FON (Fig. 2A). pH 6.4
was selected as the optimum pH for further experiments giving
short analysis time with good resolution. In addition, FAM has a
higher stability at this pH according to Junnarkar and Stavchansky
study [12].
(4)
The influence of % ACN in the mobile phase was also studied
over the range of 40–90 %, adjusting the final pH of the mobile
phase to 6.4 in each experiment. From the retention profile curve
of the two analyte as a function in ACN content in the mobile phase
(%, v/v) (Fig. 2B), it is noted that the retention of both compounds is
scarcely varied with increasing the content of ACN up to 80 %, v/v.
Even though, both FAM and FON are adequately retained on the
HILIC column under these conditions. The retention of the two com-
pounds increased by using 90 % ACN, and it was observed that the
peak shape was adversely affected and broadened. This is probably
due to the greater hydrophilic interactions and stronger retention
at high ACN ratio [1,7]. Since satisfactory resolution and adequate
retention together with symmetric and sharp peaks were achieved
at lower ACN content, 60 %, v/v ACN was chosen as the optimum
for consequent studies.
Then, the effect of ammonium acetate concentration on the
retention and separation of FAM and FON was executed over the
range of 0.01−0.1 M. In the HILIC mode, the salt concentration
directly influences the ionic interaction, where higher salt concen-
tration suppress the ionic interaction. Experiments showed that,
the retention of FON decreased significantly with the increase
in salt concentration, while FAM retention was slightly affected.
The dependence of the analytes retention on the concentration of
mobile phase counter ion can be demonstrated as follows [1,27]:
Logk = Constant–S log[C] (5)
Where; k’ is the capacity factor, [C] is the concentration of the salt in
the mobile phase, the Constant includes the phase ratio, the IE equi-
librium constant, and IE capacity of the stationary phase, and S is the
ratio of the charge of solute ion and counter ion (salt) in the mobile
phase. It is anticipated that a 1:1 complex is formed between the
counter ion (acetate) and the chemically-bonded charged groups on
the column surface (positive triazole group) [26] (Fig. 1A). As the
(3) slope (S) becomes closer to -1, this means that IE dominates where
almost complete exchange between the solute and the counter ion
takes place, while S value closer to zero means weak IE.
Plots of Log k’ versus log [C] were graphed for FAM and FON and
a linear correlation was observed (Fig. 2C). The S value for FAM was
+0.14 while it was -0.61 for FON. The positive value of the slope for
FAM is a suggestive of a net electrostatic repulsion with the station-
ary phase due to similar charges, which explains its steady tR with
different concentrations of the salt. On the other hand, the value
of the slope for FON points to synergetic combination of IE and
partitioning mechanisms, thus it showed a variable tR with chang-
 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305
Fig. 2. Retention profiles of FAM and FON on HILIC column as a function of (A) pH, and (B) %ACN in the mobile phase, (C) log k’ versus log buffer concentration [C] curve for
HILIC separation of FAM and FON, retention profiles of FAM and FON on PBr column as a function of (D) pH and (E) %ACN in the mobile phase.
ing the salt concentration. 0.05 M ammonium acetate was selected
for further study since it gave a good resolution in a reasonable
analysis time. Fig. 1A illustrates the mechanism of interaction and
separation of the two analytes by the HILIC column.
The flow rate of the mobile phase was also considered by varying
it from 0.6 to 1.4 by 0.2 mL/min increments. 1.0 mL/min was taken
as the optimum flow rate yielding good resolution and analysis time
(5 min). A typical chromatogram depicting the separation of FAM
and FON using a mobile phase composed of ACN: 0.05 M ammo-
nium acetate (60:40, v/v) of pH 6.4 at flow rate of 1.0 mL/min and
the Cosmosil® HILIC column is presented in Fig. 3A.
3.2. RPLC separation of FAM and FON on
pentabromobenzyl-bonded silica column
Initial studies on PBr column involved the effect of pH of ammo-
nium acetate buffer on the retention and resolution of the two
analytes. Preliminary, the pH study was conducted using a mobile
phase containing 30 %, v/v ACN, but early co-elution of FAM and
FON at 2.1 min was observed. So, consequent investigations were
done while keeping ACN ratio at 15 %, v/v. As seen in Fig. 2D, a
remarkable dependence of the two solutes retention on the pH is
apparent. The retention of FAM decreased by the decrease of the
pH while the retention of FON increased and a complete co-elution
of the two compounds took place at pH 5.3. The protonation of FAM
increased by decreasing the pH (Supplementary material, Table
S1), so it is eluted earlier due to the increased polarity, and vice
versa, which is a typical RP behavior. In the meantime, the carbonyl
group of FON is protonated in the acidic pH and the protonated
form is mostly resonance-stabilized (Eq. (4)) [26]. Since compounds
with delocalized electrons and resonance have greater dispersion
forces [28], the protonated form of FON exhibits stronger interac-
tion with the PBr stationary phase exhibiting longer retention at
lower pH. Despite the mobile phase of pH 5.7 gave shorter analysis
time, mobile phase of pH 6.3 was finally selected as the optimum
to preserve the maximum stability of FAM.
Secondly, the influence of ACN ratio on the retention of the
two compounds was examined. The two solutes showed a typi-
5
cal RP behavior, where their retention decreased with the increase
of %ACN (Fig. 2E). 25 %ACN was considered the optimum vol-
ume to give sharp symmetrical peaks and good resolution in a
short run time. The concentration of ammonium acetate buffer
was also studied from 0.005 M to 0.05 M. The increase in buffer
concentration leads to a slight decrease in the retention of FAM
with a minor effect on FON. Therefore, we chosen to use 0.01 M
ammonium acetate buffer for maintenance of the column durabil-
ity. Then, the flow rate of the mobile phase was studied from 0.6
to 1.4 mL/min, and 1 mL/min was finally selected to be the opti-
mum.
As a result of these studies, the optimum mobile phase for best
separation on PBr column is a mixture of ACN and 0.01 M ammo-
nium acetate buffer (25:75, v/v) of pH 6.3 at a flow rate of 1 mL/min.
Fig. 3B shows a typical chromatogram for separation of FAM and
FON under these conditions.
Fig. 1B illustrates the mechanism of interaction and separation
of the two candidates by the PBr column. The elution order on PBr
column, FON then FAM, under the optimum analytical conditions
can be explained according to the following concept:
i Common interaction forces exist between the two compounds
and the stationary phase include: ␲ stacking between the aro-
matic rings of the two compounds and the stationary phase, ␲-␲
interactions between the aliphatic residues of the two compounds
and the aromatic ring of the stationary phase [29].
ii Cation–␲ interaction occurs between cationic FAM and the aro-
matic ring of the stationary phase [29].
iii Ion–induced dipole interaction between cationic FAM and the
brominated stationary phase [30].
iv Dipole-induced dipole interaction between FON carbonyl group
and the brominated stationary phase [30].
v Since ion–induced dipole interaction is stronger than
dipole–induced dipole interaction as the charge of any ion is
far greater than the charge of a dipole moment [30], in addition to
the cation–␲ interaction associated with FAM only, FAM exhibits
stronger binding to the stationary phase than FON and eluted
later.
6 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305
Fig. 3. Representative chromatograms obtained after the incubation of FAM
(15.0 ␮g/mL) at 37 ◦ C for 30 min in 0.06 M HCl showing the separation of FAM and
FON using (A) HILIC and (B) RPLC modes, where (a) is FAM and (b) is FON.
3.3. Criteria for selection of the most proper separation mode
The selection of the better separation mode is judged with
three criteria in mind: (i) the chromatographic performance, (ii)
the method harmony with green chemistry concept, and (iii) the
economy character.
Table 1 shows a comparison of the chromatographic per-
formance of the two separation modes under their optimum
conditions. We can realize that the analysis time in case of PBr
column is slightly shorter than the HILIC column, meanwhile, the
peak areas are quite similar. As well, the HILIC column has a rather
higher number of theoretical plates (NTP) for FAM and FON while
the PBr column has a smaller height equivalent to the theoretical
plate (H) for both compounds. The higher NTP in case of HILIC col-
umn is explained by being longer than the PBr one (250 mm versus
150 mm, respectively). Despite that, the H values for the PBr column
are approximately 1.5 times lower than those for the HILIC column
which indicates the greater efficiency of the PBr column relative to
its length. For a good LC practice, it is better to minimize the extra-
column volume especially if no additional advantage is gained. The
resolution factors were further evaluated [13]. As deduced from
Fig. 3, the two LC modes excellently resolved the two analytes, with
the higher resolution (Table 1) and superior peak shape provided
by the RPLC method.
Overall consideration of these entire factors revealed compara-
ble chromatographic performance of the two LC modes, with some
surplus advantages for the RPLC method regarding shorter analy-
sis time and usage of a shorter column. In addition, the successful
application of the HILIC mode needs a special attention to ensure
adequate column conditioning before use and re-equilibration
between injections since insufficiency of these steps can result in
failure in re-establishing the aqueous layer on the particles of the
packing which causes fluctuation in tR and irreproducible results.
From the aforementioned perspectives; we, so far, outweigh the
RPLC method over the HILIC method.
For assessing the greenness of the developed method and its
influences on health, safety, and environment, a three-sided green-
ness assessment approach was adopted for a true judgment. First,
we used the Green Analytical Procedure Index (GAPI) [31] that is
based on 5-pentagrams representing different stages of the ana-
lytical procedure. The GAPI pictograms for the two LC methods is
shown in Table 2 and their interpretation is shown in Table S2 (Sup-
plementary material). The obtained results showed that, the green
character of the two methods are similar. The second metric to
evaluate the greenness of the two studied LC modes is the ana-
lytical eco-scale [32] that considers the nature and the volume of
reagents, instruments used, energy consumed, occupational haz-
ards, and volume of waste. Penalty points are given to each factor,
and the sum of penalty points is subtracted from 100 as the score for
ideally green method. The two LC methods had the same analyti-
cal eco-scale score of 76 which means excellent greenness (Table 2
and Supplementary material Table S3). The similar responses in
these two metrics are attributed to giving the same impact to a
wide range of reagent volumes, where the analytical eco-scale score
approach gives a penalty point of 2 to any reagent volume ranged
from 10 to 100 mL [32] and the GAPI approach assigned a red color
for any reagent volume > 10 mL [31]. Thus, despite the RPLC method
employed a mobile phase containing much lower amount of ACN
than the HILIC method, the two metrics could not discriminate the
two modes.
Thus, the HPLC-Environment Assessment Tool (HPLC-EAT) [22]
was utilized to mainly evaluate the impact of the solvents used in
the HPLC method. The HILIC method’s EAT unit is 10.731 while that
of the RPLC method is 3.576 (Table 2). The higher EAT unit of the
HILIC method is attributed to using a higher concentration of ACN
in the mobile phase. Thus, the RPLC method is considered greener
than the HILIC method with lower safety, health, and environmen-
tal impacts (Supplementary material, Fig. S1).
In term of cost efficiency, the RPLC method consumes less
amount of acetonitrile for the mobile phase, in addition to using
a shorter column (150 mm) that needs smaller volumes of organic
solvent for washing and equilibration with the mobile phase before
starting the analysis. Hence, the RPLC method is considered more
economic and cost-effective.
From the above-mentioned results it is concluded that the RPLC
method is superior to the HILIC method in terms of chromato-
graphic performance, method greenness, and cost-effectiveness.
Despite the difference is not very intense, the RPLC method was
the method of choice for studying the gastric stability of FAM and
the next validation and application steps are done adopting the
RPLC method.
3.4. Validation of the developed method
The developed RPLC method was validated according to the
Food and Drug Administration (FDA) instructions [33] using Solu-
 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305 7

Table 1
Chromatographic performance of HILIC and RPLC modes for separation of FAM and FON.

Retention time (tR ±SD)a Peak area Number of theoretical Height equivalent Resolution
Method
plates (NTP)b theoretical plates (H, mm)c (Rs )d

FAM FON FAM FON FAM FON FAM FON

HILIC 3.14 ± 0.05 4.77 ± 0.17 66,586 82,776 8589 12,605 0.029 0.020 6.0
RPLC 3.89 ± 0.04 2.48 ± 0.14 69,295 82,305 7790 12,030 0.019 0.012 9.9
a
Average of ten injections.
b
NTP = 5.54(tR /w1/2 )2 .
c
H = L/NTP.
d
Rs = 2(tR2 -tR1 )/W1 +W2 .

Table 2
Comparison of HILIC and RPLC methods regarding greenness and eco-friendship using different evaluation metrics.

Evaluation metric HILIC RPLC

Analytical eco-scale score 76 76

HPLC-EAT units 10.731 3.576

GAPI

Detailed calculations of analytical eco-scale scores, interpretations of references to colors in GAPI pictograms and detailed impacts of HPLC-EAT are provided in the
supplementary materials.

Table 3 a considerable UV absorption at the detection wavelength, 267 nm

Linearity, accuracy, and precision data for the developed RPLC method.
[35]. This fact is supported by the absence of such peak in chro-
Linearity matograms of FAM treated with HCl only instead of SGJ (Figs. 3B
and 4C).
Parameter Results
Moreover, the accuracy and precision of the RPLC method
Concentration range (␮g/mL) 1.0−20.0
were estimated using three concentrations of FAM (1.0, 8.0, and
Limit of detection (LOD) (␮g/mL) 0.14
Limit of quantification (LOQ) (␮g/mL) 0.38 20.0 ␮g/mL for low, medium, and high concentration) and 5 repli-
Correlation coefficient (r) 0.9998 cate per concentration in the same day (intraday level) or on 5
Slope 8.567 × 103 respective days (interday level) (Table 3). The %RSD for intra- and
Intercept 1.245 × 103 inter-day precision was ≤ 2.38 indicating the high precision of the
Standard deviation of the residuals (Sy/x ) 1.08 × 103
Standard deviation of the intercept (Sa ) 3.23 × 102
developed method. As well, the intra-day and inter-day accuracy of
Standard deviation of the slope (Sb ) 62.87 the developed RPLC method was also checked using the same three
% RSD 2.87 concentrations and it ranged from 98.70 to 100.67% (Table 3). These
√
% Error (% RSD/ n) 1.08 values confirmed the accuracy of the developed method.
The stability of stock solution and resolution solution was also
Accuracy and precision
examined by comparing the response of fresh solutions with aged
Conc. Intra-day Inter-day solutions maintained for 5 days in the refrigerator at 4 ◦ C. As well,
(␮g/mL) the bench-top stability of the samples, prepared according to proce-
Accuracy Precision Accuracy Precision
(%Found) (%RSD) (%Found) (%RSD) dure in “Section 2.6”, was also checked up to 6 h under laboratory
handling conditions at 20 ◦ C. Excellent stability was achieved in
1.00 98.70 2.01 100.67 2.38
8.00 99.34 2.31 99.26 0.99 both levels where the accuracy was within +0.67 to −0.74 % and
20.00 99.91 1.35 99.35 0.92 −0.09 to −1.30 % of the nominal concentrations, respectively.
From the aforementioned results, we can conclude that the
developed RPLC method met the acceptance criteria for FDA bio-
analytical method validation [33].
tion A (pH 3.5) as a modified SGJ to overcome the instability of FAM
in SGJ. Calibration curve based on the peak area showed excellent
linearity over the range of 1.0−20.0 ␮g/mL with a correlation coef- 3.5. Comparison of the proposed method and the reported
ficient r = 0.9998. The limits of quantification and detection were literature for separation of FAM and FON
found to be 0.38 and 0.12 ␮g/mL, respectively. The linearity data
are collected in Table 3. As compared to the reported HPLC methods for separation of
The method selectivity is revealed by the freedom of the blank FAM and FON [8, 13, 36–38], the proposed method is characterized
from interference from SGJ components at the retention times of by its rapidness, high samples throughputs, ideal chromatographic
the two analytes. Additionally, no significant matrix effect was peaks, simple mobile phase, and isocratic elution. The reported
observed from SGJ components on FAM, where the statistical com- HPLC procedures either necessitate gradient elution [8,38], water-
parison of the assay results in Solution A and in the mobile phase rich mobile phase [13,37], ion-pairing additive-containing mobile
by t- and F-tests showed no significant difference since the calcu- phase [8,36], or temperature-controlling [8,13,38]. Additionally,
lated t- and F-values were < the theoretical values [34] (Table 4). the long analysis time (≈ 25 min) [8,37] and the poor sensitivity [37]
However, the small peak appeared ahead of the chromatograms are main disadvantages of some of the reported methods. Table 5
obtained for SGJ experiments (Fig. 4) is assigned for pepsin that has displays a comparison of the performance of the proposed method
8 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305

Table 4
Statistical evidence of non-significant matrix effect on FAM in SGJ (pH 3.5) relative to in the mobile phase.

Matrix
Parameter
Mobile phase SGJ (pH 3.5)
a
Conc. taken (␮g/mL) Conc. found (␮g/mL) % Found Conc. taken (␮g/mL) Conc. taken (␮g/mL) %Founda

1.00 0.986 98.60 1.00 0.987 98.70

8.00 7.989 99.86 8.00 7.947 99.34
20.00 20.034 100.17 20.00 19.981 99.91
Mean ± SD 99.54 ± 0.83 99.32 ± 0.61
Variance ratio F-testb 1.89 (19.00)
Student’s t-testb 0.38 (2.78)
a
Each result is the mean of 5 separate determinations.
b
The numbers in parentheses are the theoretical t and F-values [34].

Fig. 4. Representative chromatograms showing FAM (10.0 ␮g/mL) after incubation for 1 h at 37 ◦ C in (A) SGJ of pH 3.5, (B) SGJ of pH 1.2, (C) 0.06 M HCl, pH 1.2, (D) SGJ of pH
1.6, and (E) SGJ of pH 2.0, where (a) is pepsin peak, (b) FAM, and (c) FON at respective tR = 1.29 ± 0.03, 3.89 ± 0.04, and 2.48 ± 0.14 min.

and the published literature for separation of FAM and FON showing
the main merits of the new method.
3.6. Study of FAM stability in SGJ
The investigation of drug stability in gastric juice is important
for determining if the drug is lost due to poor intestinal perme-
ability or the degradation in such medium has an impact prior to
membrane absorption. The Food and Drug Administration (FDA)
guidance on in vivo Bioavailability and Bioequivalence Studies for
Immediate-Release Solid Oral Dosage Forms [19] recommended
to study the drug stability using simulated fluid since taking real
fluids from human is problematic and needs intubation, instead,
USP fluids [13] can be used. Some simple steps were addressed by
the FDA as a guidance for conducting such studies by incubation
in the simulated fluid at 37 ◦ C for a suitable time period similar
to the actual contact time of the drug with the fluid. Degradation
of more than 5% of the drug is indicative of a potential instability
[19].
FAM is known by its limited oral bioavailability since only 20–66
% of the oral dose gets systematically available in healthy humans.
All the oral dosage forms of FAM (suspension, tablets and cap-
sules) have the same bioavailability [9], which indicates that the
limited bioavailability is not a dosage form-related issue. Yet, the
exact reason of this poor bioavailability is not completely known. It
is not clear whether FAM is lost by degradation in the stomach
prior absorption or the sole reason for its limited bioavailabil-
ity is the low intestinal permeability. Since the developed RPLC
method is stability-indicating and it is able to resolve FAM and
its acidic degradation product FON, it was applied for the inspec-
tion of FAM stability in SGJ to give indication about the degree
of its decomposition in the stomach prior intestinal permeation
process.
 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305 9

Table 5
Comparison of the performance of the proposed method and the published literature for separation of FAM and FON.

Method Conditions Column LOD (␮g/mL) Analysis time Remarks Ref.

(min)

HPLC-UV Mobile phase A: mixture of C18 – ≈25 • IPC, high column [8]
methanol: ACN: sodium temperature, and gradient
hexanesulfonate pH 3.5 (6:94:900, elution are needed.
v/v/v), mobile phase B: ACN, • Long analysis time
temperature: 50 ◦ C
HPLC-UV Mobile phase is a mixture of C18 – – • High water content in the [13]
acetate buffer (pH 6.0): ACN (93:7, mobile phase and high
v/v), flow rate 1.4 mL/min, UV column temperature are
detection at 275 nm. needed.

HPLC-UV Mobile phase is 50:50 v/v ACN: Porous 0.1 ≈6 • IPC is needed. [36]
water containing 0.5% pentane graphitic • Poor resolution of FAM and
sulphonic acid, UV detection at carbon FON
265 nm.
HPLC-UV Mobile phase is a mixture of C18 0.4 ≈ 25 • Long analysis time [37]
ammonium acetate buffer (pH 2.9)
and ACN (84: 16, v/v), flow rate of
1.5 mL/min, UV detection at
254 nm.
UPLC-UV Mobile phase A: a mixture of BEH Shield C18 6.0 ≈2 • Gradient elution and [38]
sodium acetate buffer (pH 6.0): temperature control are
methanol (80:20, v/v), mobile needed.
phase B: a mixture of sodium • Poor sensitivity.
acetate buffer (pH 6.0): methanol
(30:70, v/v), UV detection at
260 nm, temperature: 30 ◦ C
HPLC-UV Mobile phase is a mixture of ACN PBr 0.14 ≈4 • Isocratic elution with Present
and 0.01 M ammonium acetate simple mobile phase. method
buffer (25:75, v/v) of pH 6.3, flow • Short analysis time and high
rate of 1 mL/min, UV detection at sensitivity.
267 nm. • Ideal peaks and excellent
resolution.

With this goal, FAM (20.0 mg) was incubated in 50 mL of USP Table 6
Kinetic parameters for the degradation of FAM under different conditions.
SGJ (pH 1.2) [13] at 37 ◦ C and the degradation was monitored by
the developed RPLC method up to 160 min. The quantity of FAM in %Degradation Reaction rate Half-life
Condition constant, k time, t1/2
the test is based on the available oral dosage forms (20 and 40 mg
60 min 160 min (min−1 ) (min)
tablets and 40 mg/5 mL oral suspension). As well, the average vol-
SGJ, pH 1.2 43.2 74.8 8.1 × 10−3 86
ume of gastric juice is about 50 mL [39], so we used this volume
0.06 M HCl, pH 1.2 32.2 60.6 5.0 × 10−3 138.6
to conduct the present study. Despite the FDA [19] suggested to SGJ, pH 1.6 14.0 15.0 4.6 × 10−4 1506
study the gastric stability for 1 h, we extended the investigation to SGJ, pH 2.0 11.5 14.1 3.92 × 10−4 1767
160 min since the gastric emptying time may vary from 2−3 hrs in
response to many factors [40].
The obtained results confirmed the high instability of FAM under (Table 6) confirming the pH-dependent degradation of FAM. Fig. 4D
this condition compared to the control (Fig. 4A). A gradual degra- and E illustrate chromatograms for FAM in SGJ of pH 1.6, and 2.0,
dation of FAM with time was observed where about 43.2 % of the respectively.
drug degraded after 1 h yielding FON (Fig. 4B). A kinetic follow-up This investigation highlights the instability of FAM in the stom-
of the degradation process revealed that it is a pseudo-first order ach and suggests that gastric degradation is a major player in the
reaction in HCl concentration. The reaction rate constant was calcu- drug loss and its poor bioavailability rather than the intestinal per-
lated and was found to be 8.1 × 10−3 min-1 . To understand whether meation.
this degradation behavior is driven by the pH only or it is promoted
by the gastric enzyme and other components, we conducted a study 3.7. Comparative molecular docking study of FAM and FON at
of FAM degradation in 0.06 M HCl (pH 1.2) omitting the other con- hH2 R receptor and ADMET prediction
stituents of SGJ [13]. We found that, the drug is also highly unstable
in this condition following a pseudo-first order degradation. Yet, The previous study of Yanagisawa et al. [41] suggested that
approximately 32.2 % degradation was detected after 1 h (Fig. 4C), FAM outperforms FON H2 -antagonist activity, however this was
and the reaction rate constant was 5.0 × 10-3 min-1 . Thus, it is con- a hypothesis and their interaction pattern into hH2R recep-
cluded that the degradation of FAM is mainly pH-dependent, but tors has not been compared yet. hH2 R is a member of the G
the components of SGJ probably have a catalytic effect on the degra- protein-coupled receptor (GPCR) group that is characterized by 7
dation reaction that contributes to the higher % degradation and transmembrane segments (TM1–TM7). Since hH2 R has not been
reaction rate constant in SGJ (Table 6). crystalized yet, we utilized its homology model as an alternate
Furthermore, for emphasizing the effect of pH on the degra- tactic [42] as per our previous study [43]. In this context, GPCR-
dation of FAM, and since the gastric pH may vary from 1.0 to 2.0 turkey ␤1 -adrenoreceptor was utilized as a prototype to generate
[40], we studied the degradation kinetically in modified SGJs of hH2 R model by virtue of the remarkable amino acid resemblance
pH 1.6 and 2.0. A dramatic decrease in the %degradation and the (≈38 % sequence identity) [44]. After creation of 10 models,
reaction rate constants together with increased t1/2 were obvious the most suitable one was chosen based on Ramachandran plot
10 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305

Fig. 5. Binding mode of (A) FAM green sticks and (B) FON blue sticks, into hH2 R model’s pocket. Key amino acid residues are depicted in Simon sticks. All atoms, other than
carbons, were colored by element and settled intermolecular interactions were shown as black dashes. (C) and (D) are 2D-depiction of FAM and FON, respectively, into the
hH2 R model’s pocket (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.).

(Supplementary material, Fig. S2A). The model did not display out-
lier residues or atom clashes (Supplementary material, Fig. S2B)
approving its rationality for the following modelling steps. Dock-
ing of FAM into hH2R model pocket revealed that its terminal
guanidine forges a salt bridge with Asp98 side chain in accor-
dance with a previous similar docking report [45]. Mutagenesis
study on hH2R has established Asp98 at transmembrane 3 (TM3)
to be essential for histamine binding [46,47]. Furthermore, the
N-́sulfamoylformimidamide group of FAM forms three interactions
with Asp186 and Tyr250 which anchors FAM inside the active site
with a binding energy of -7.8 kcal/mol (Fig. 5A and C). Although FON
keeps binding Asp186 and Tyr250, it lacks the key interaction with
Asp98 and its binding energy is -5.7 kcal/mol (Fig. 5B and D). The
reduced activity of FON compared to FAM seems to be attributed
to missing a crucial stabilizing interaction with the key Asp98.
Then we tried to get more insights into FON toxicity/safety pro-
file. In silico toxicity prediction showed that the gastric degradation
does not appear to cause additional toxicity. Instead, FON seems to
be relatively safer than FAM itself in some aspects. According to
the ADMET toxicity prediction module [24], FAM is associated with
an elevated level of serum alkaline phosphatase (Ser AlkPhos), a
mutagenic activity in one of the 10 tested bacterial strain models,
and a greater rat and mouse toxicity than FON (Table 7), while these
effects are not accompanying FON. On the other hand, no endocrine,
cardiac, or reproductive toxicity is detected for both compounds. As
well, both drugs cannot induce chromosomal aberration or phos-
pholipidosis [24].
The predicted metabolic profiles of the two candidates shows
also some differences with respect to the metabolizing enzymes as
well as their potential as enzyme inhibitors as obvious in Table 7.
Some differences between the two compounds were also detected
by the ADMET predictor’s PCB module regarding their solubility and
partitioning (Table 7). In addition, the pharmacokinetics of the two
compounds differs to some extent. The % of FAM unbound to human
and rat plasma protein is greater than FON which means that the
fraction of free active FAM that can cross membranes, metabolized,
and excreted is greater than FON. This contributes to different bio-
logical half-lives and clearance for the two compounds [48]. In the
same vein, the volume of distribution (the volume needed to con-
sistently distribute a quantity of a drug to achieve the observed
blood concentration, Vd) of FAM is 2 times that of FON which is
attributed to the greater plasma protein binding of FON. The human
blood/plasma concentration ratio of FAM is about 1.6 times that of
FON pointing to greater distribution of FAM to erythrocytes specif-
ically [24].
 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305 11

Table 7
Differences in the ADMET properties of FAM and FON.

ADMET property Descriptor Descriptor definition FAM FON

Toxicity module
Hepatotoxicity Ser AlkPhos Serum alkaline phosphatase level Elevated Normal
Genotoxicity MUT m102+wp2 Estimation of mutagenicity in 102-strain of S. Typhimurium and -wp2 strain of Positive Negative
E. Coli
Animal toxicity models
Rat Acute The quantity of the compound (mg/kg) that leads to killing of 50 % of the rats 621.749 3862.965
when given orally regardless of the mode of action
Rat TD50 The quantity of the compound (mg/kg) given orally to rats or mice during 17.842 25.107
Mouse TD50 their lifetimes that induces tumors in 50 % of population 12.676 18.866
Metabolism module
CYP inhibition CYP3A4 Ki testo Quantitative estimate of the substrate-specific inhibition constant (Ki) values 15.996 8.798
(␮M) in recombinant-expressed CYP3A4 with testosterone as substrates
CYP2C9 Inh Classification of drugs as inhibitors/non-inhibitors against CYP450 Yes No
CYP1A2 Inh isoforms Yes No
UGT substrate UGT1A9 No Yes
Classification of drugs as being metabolized (yes) or non-metabolized (no)
UGT1A4 Yes No
by UGT isozymes
UGT2B7 Yes Yes
ADMET Predictor’s
PCB Module
pKa S + Basic pKa* Estimation of pKa based on a thermodynamically accurate multiprotic model 6.67 6.86
for multiple ionization sites according to atomic descriptors and neural
networks not a database.
Partitioning S + logP Quantitative estimate of the partition coefficient between octanol and water. −0.498 −1.172
S + logD Quantitative estimate of the distribution coefficient between octanol and −0.572 −1.096
buffered water.
Pharmacokinetics
hum fup% %Unbound of the drug to human plasma protein 58.32 37.727
rat fup% %Unbound of the drug to rat plasma protein 37.467 26.831
Vd Human volume of distribution 1.199 0.549
RBP Human blood/plasma concentration ratio 1.708 1.018
RBP rat Rat blood/plasma concentration ratio 0.876 0.705
Solubility models S + FaSSGF Solubility in fasted state SGJ 3.71 6.187
S + FaSSIF Solubility in fasted state simulated intestinal fluid 1.426 7.339
S + FeSSIF Solubility in fed state simulated intestinal fluid 0.361 0.626
*
The strongest basic pKa .

3.8. Recommendations for practices to optimize FAM activity and
safety
On the light of these findings, we can recommend in-situ
buffered formulation to increase the stability of FAM in the stomach
and improve its bioavailability. In this tactic, the formulation con-
tains a substance that buffers the site of drug delivery then release
the drug in a non-acidic medium, thus increasing its stability [49].
Moreover, the in-situ buffering will enhance the solubility of FAM
since it is a weakly basic drug [50]. Since FAM acts by competitive
binding to the H2 -receptors in the parietal cells present in the gas-
tric glands [51], the enteric-coating is not an applicable solution for
the problem of FAM instability where it prevents the drug release
in the stomach and allowed its release in the intestine. This means
that enteric coating leads to loss of FAM activity. On the other hand,
the results of the in silico studies revealed the necessity to conduct
a safety lead optimization of FAM to minimize the potential toxic
properties and side effects while maintaining its activity at hH2 R.
The outcomes of this study are important not only for the
widespread use of FAM as anti-ulcer, but also due to the expected
future increase of its applications as adjunct therapy for patients
with resistant schizophrenia [52].
while the two techniques have comparable chromatographic per-
formance. Based on the obtained results, the RPLC mode using
the new PBr column was selected for LC separation of the two
analytes. The developed RPLC method met the acceptance crite-
ria of FDA on bioanalytical method validation. Application of the
developed method to study the stability of FAM in SGJ revealed
its high vulnerability to degradation under the intra-gastric con-
ditions where about 43.2 % of the drug degraded after 1 h. The
pseudo-first order degradation reaction was proved to be pH-
dependent and catalyzed by the gastric juice. This degradation
probably has a significant role in the poorer oral bioavailability
of FAM. Molecular docking study evidenced that FON lacks a cru-
cial stabilizing interaction with the key Asp98, leading to poor
activity as H2 -antagonist. However, some differences in the toxi-
city profiles of FAM and FON were also detected in favor of FON.
Moreover, differences in the pharmacokinetics, metabolism, and
solubility of the two compounds were also identified using the
ADMET prediction tool. We suggest in-situ buffered formulation
for protection of FAM from gastric degradation. As well, safety lead
optimization of FAM is suggested to curtail its potential toxic side
effects.

3.9. Conclusions CRediT authorship contribution statement

In this contribution, a comparative study between HILIC and
RPLC modes employing newly manufactured columns was con-
ducted for separation of FAM and its acidic degradation product
FON. The RPLC method is greener with lower impacts on the safety,
environment, and health as deduced by HPLC-EAT tool. Also, less
costs and shorter analysis time is achieved by the RPLC method,
Rania El-Shaheny: Conceptualization, Methodology, Software,
Validation, Formal analysis, Investigation, Data curation, Writing
- original draft, Writing - review & editing, Visualization, Project
administration. Mohamed O. Radwan: Software, Data curation,
Writing - review & editing. Fathalla Belal: Writing - review & edit-
ing. Koji Yamada: Funding acquisition, Resources.
12 R. El-Shaheny, M.O. Radwan, F. Belal et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113305
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper.
Acknowledgements
This work was supported in part by a Grant-in-Aid for Sci-
entific Research No. 23590008, 26460124, and 18K06718 from
the Japan Society for the Promotion of Science (JSPS), which is
gratefully acknowledged. R.E. is thankful to The Takeda Science
Foundation (Osaka, Japan) for providing the fellowship in Nagasaki
University, Japan. The authors are grateful to Prof. Masami Otsuka
from Kumamoto University and the founder of Science Farm Ltd.,
Kumamoto, for his support in performing the in silico study.
Appendix A. Supplementary data
Supplementary material related to this article can be found,
in the online version, at doi:https://doi.org/10.1016/j.jpba.2020.
113305.
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