Journal of Chromatography B 1166 (2021) 122549

Contents lists available at ScienceDirect

Journal of Chromatography B
journal homepage: www.elsevier.com/locate/jchromb

In depth investigation of the retention behavior of structurally related

β-blockers on RP-HPLC column: Quality by design and quantitative
structure-property relationship complementary approaches for
optimization and validation
Ahmed Faried Abdel Hakiem a, *, Ahmed K. Hamdy b, Hassan Refat Hassan Ali c,
Mohamed Gomaa d, Ahmed Safwat Aboraia b
a
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh 33516, Egypt
b
Medicinal Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
c
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
d
Botany and Microbiology Department, Faculty of Science, Assiut University, Assiut 71516, Egypt

A R T I C L E I N F O A B S T R A C T

Keywords: The persistent introduction of new β-blockers motivates the demand for optimizing RP-HPLC well-designed
β-blockers analytical procedures that could be applied to this structurally related and commonly prescribed pharmaco­
Betoxolol HCl and carvidolol logical group in order to reduce time and chemicals consumption in quality control units. Betoxolol HCl (BEX)
RP-HPLC
and Carvidolol (CAR) were selected as representative examples to conduct predictive studies based on two
Quality by design and quantitative structure
property relationship
complementary approaches, Quality by design (QBD) and Quantitative structure property relationship (QSPR).
Derringer’s desirability function In concern QBD, a Box-Behnken design was adopted at variable chromatographic parameters to achieve the most
ICH validation guidelines proper conditions that might be applied for efficient analysis of the majority of group members. On the other
hand, the retention time was chosen as the target property in the QSPR study that was conducted onto seven β.
blockers (the two investigated drugs in addition to five other β. blockers) to find the best correlated molecular
descriptors to the retention behavior. Both external and internal validation studies have comparable quality with
training levels. Hence a simple selection algorithm of conventional features provides robust confirmatory pre­
dictive QBD and QSPR models. Derringer’s desirability function as as a multi-criteria approach was applied for
getting the optimum chromatographic analysis conditions. Efficient analysis of BET and CAR was achieved at
column temperatures of 26.00 and 27.50 ◦ C, respectively using acetonitrile and phosphate buffer (pH 4.55)
70:30 v/v as a mobile phase with a flow rate of 1.00 mL/min, and UV detection at 220 nm. The method was
validated in accordance to ICH guidelines, and had exhibited acceptable precision, accuracy, linearity, and
robustness.

1. Introduction
Quality by design is defined in the i international conference on
harmonization (ICH) Q8 guidelines as a systematic approach to devel­
opment that begins with predefined objectives and emphasizes product
and process understanding and process control, based on sound science.
The key step for development of QBD is to control some chemical,
physical, microbiological or biological properties known as the critical
quality attributes (CQA) to be within an appropriate limits to confirm
the desired quality (i.e. the desired responses) [1]. It determines the
aims of the method and ensures thorough evaluations and makes
reconnaissance of alternative procedures in a systematic way to get the
optimized performance [2]. Focusing on QBD concept in analytical
methods has an increased integration within pharmaceutical process
control strategies [3,4] and the traditional pharmaceutical analysis is
not immune against deviation of specification results and statistical
control. At this juncture, implementation of QBD is considered as
mandatory by Europe Medicines Agency (EMA) as well as new drug
applications based on analytical QBD are recently approved by FDA [5].
The input variables that have represented to provide the desired quality

* Corresponding author.
E-mail address: ahmed_faried@pharm.kfs.edu.eg (A.F.A. Hakiem).

https://doi.org/10.1016/j.jchromb.2021.122549
Received 22 August 2020; Received in revised form 3 January 2021; Accepted 9 January 2021
Available online 20 January 2021
1570-0232/© 2021 Elsevier B.V. All rights reserved.
A.F.A. Hakiem et al.
Fig. 1. Chemical structures of the investigated drugs.
exhibit multidimensional interaction giving what is called the design
space. The process development is directly related to the size of this
window, in other words, if the design space is small, the process will also
need to be controlled in very narrow operating ranges to ensure that the
product meets specifications, and vice versa means that the process is
flexible [6].
The critical quality attributes that directly affect performance of
chromatographic systems should be identified firstly which are typically
sampling, sample preparation, standards, reagents, column chemistry,
mobile phase composition, pH and flow of mobile phase, column tem­
perature, detector selection, etc. These parameters affect what is called
the critical method parameters (CMPs) that can be divided into three
categories, parameters regarding instrument, parameters regarding an­
alyte and those regarding operation conditions. These parameters are
usually expressed in chromatography as system suitability like resolu­
tion, retention time, tailing factor, detection limit, robustness, etc. [7].
The majority of chromatographic analyses have been conducted by one
factor at a time (OFAT) approach. This approach requires changing a
single system parameter while the others are kept constant, resulting in
many optimization and validation runs. Instead, the chemometric
experimental design (CED) uses multivariate statistical techniques, is
considered advantageous in the reduction of experiments and hence
solvent consumption and laboratory work . In liquid chromatography,
the main determinants of analyte distribution between stationary phase
and eluent system are chemical structure of the analyte and physico­
chemical properties of both the stationary phase and the mobile phase.
The quantitative structure- property relationships approach character­
izes these criteria in numerical forms [8]. Linear free-energy relation­
ships (LFER) represents the basis of QSPR modeling, and
physicochemical properties are considered as different free energy
transducers. Therefore, different retention behaviors of analytes can be
explained in terms of differences in chemical potentials with respect to
structural differences and hence quantitative differences in the proper­
ties, such as presentation retention (the logarithmic value of the reten­
tion factor, log k) [9]. Retardation factors were considered as the target
propert ies to establish QS PR models. These values are then subjected to
chemometric processing by partial least square (PLS) analysis to build
up correlations between these properties and retention behaviors. De­
scriptors selection was performed by advanced nature-inspired firefly
algorithm [10,11]. Herein, a great number of quantitatively comparable
retention behaviors for large sets of structurally related and diversified
analytes have to be studied. Because of similarity in experimental con­
ditions, the obtained data can be mutually correlated [12]. In RP-HPLC,
interactions of analytes with the non-polar hydrocarbon chain station­
ary phase, can be identified in terms of different dispersive forces, also
polarizability differences lead to different dispersion interactions that
2
Journal of Chromatography B 1166 (2021) 122549
reflects variable molecular sizes (London-Hall effect) [13]. On the other
hand, the effect of orientation interactions (Keesom effect) is negligible,
because of the negligible polarity (dipole moment) of the hydrocarbon
part of the molecules, also the dipole-induced dipole interactions (Debye
effect) is supposed to be similar for all analytes. Analytes provide
different interactions with the polar eluent that can be explored as
stronger orientation and inductive effects as well as polarizability
dependent dispersive and inductive effects, at the same time, analytes
possess relatively similar dipole–dipole and dipole-induced dipole in­
teractions. Hence, RP. HPLC interactions can be classified as Van der
Waals interactions (Keesom, Debye, and London-Hall effects) and more
complicated ones (mainly, hydrogen bonding and charge transfer in­
teractions). These complicated interactions can be interpreted by vari­
ations in an unlimited number of structural descriptors [13]. Betaxolol
HCl and CAR Fig. 1 have been selected as representatives for this po­
tential pharmacological group in this study. To date, there are no
analytical QBD studies have been conducted involving BET and CAR.
Many QSPR reports involving CAR and BEX were conducted on hydro­
philic columns (HILIC columns) using MLR and artificial neural network
(ANN) [14,15] and CAR alone using advanced models like Kernel PLS
(KPLS) and Levenberg–Marquardt artificial neural network (L–M ANN)
by UPLC in blood samples [16]. The Quality by design approach was
applied only in supercritical chromatographic analytical studies of CAR
[17]. In concern BET, a number of QSPR studies had been applied using
HPLC technique on HILIC columns [18–22], RP HPLC [23,24], affinity
chromatography [25,26] and salting out TLC [27]. In the presented
study, the selection of BET and CAR provides an attempt to develop
simple, rapid, sensitive, robust, and economic HPLC method utilizing
analytical QBD basics for screening of beta-blockers in bulk drug and
pharmaceuticals. The rational use of experimental design was presented
for comprehensive illustration of the factor–response relation then
ensuring of robustness by validation studies. Additionally, building up
reliable QSPR models including Five other β.blockers, namely; Atenolol
(ATE), Bisoprolol fumarate (BIS), Nebivolol HCl (NEB), Labetalol HCl
(LAB) and Propranolol (PRO) for get ting accurate correlation results c
could help possible interpretation of the chromatographic behavior of β.
blockers class. This introduces prediction as well as rationalizing of the
group members even prior to synthesis depending on common structural
features. Moreover, the experimental retention behavior of the repre­
sentative analytes provides a guide for selection of the proper working
conditions as well as a facility for hitting the best descriptors describing
the chromatographic variations.
2. Materials
Atenolol, BIS, BEX, CAR, LAB, NEB and PRO were kindly provided as
A.F.A. Hakiem et al.
free gifts of a number of pharmaceutical factories working in Egypt,
AstraZeneca (6th October city, Giza, Egypt), Global Napi Pharmaceuti­
cals (6th October city, Giza), (Borg Pharmaceutical Industries, Borg El-
Arab New City, Alexandria, Egypt), (Multi-Apex Pharma, Badr City,
Cairo, Egypt), Aldbeky Pharmaceutical Industries (El-Obour city, Cairo,
Egypt), Marcyrl Pharmaceutical Industries (El-Obour city, Cairo, Egypt)
and AstraZeneca (6th October city, Giza, Egypt), respectively. Acetoni­
trile (HPLC grade) was purchased from Sigma-Aldrich (St. Luis, USA).
Ortho phosphoric acid and KH2PO4 were obtained of El Nasr company
for medicinal chemicals (Abou Zaabal - El Qalyubia – Egypt). All sol­
vents and chemicals were of analytical grade. Pharmaceutical formula­
tions were purchased from the Egyptian market; Betaxolol 20 mg®
tablets (Borg Pharmaceutical Industries, Borg El-Arab New City, Alex­
andria, Egypt) and Carvid 25 mg ® tablets (Multi-Apex Pharma, Badr
City, Cairo, Egypt).
3. Methods
3.1. Preparation of phosphate buffer
Into four 1000.00 mL volumetric flasks, weighed amounts of 0.6805
gm of KH2PO4 were dissolved in 200.00 mL double distilled water and
then completed to the mark to get 0.005 M solutions. Firstly, three
portions of 500.00 mL were transferred into beakers and then adjusted
to pH values: 3.15, 3.85 and 4.55 for test set experiments. Three other
portions each of 500.00 mL were adjusted to pH 4.00, 4.55 and 5.05 for
experimental set as well as validation experiments. Finally, 1000.00 mL
portion was adjusted to pH 4.55 for QSRR study. The prepared buffer
solutions were filtered through 0.22 μm membrane and then sonicated
for 30 min before use.
3.2. Standard solutions preparation
Accurately weighed 0.10 g amounts of studied analytes; BEX and
CAR were transferred into 100.00 mL volumetric flasks and made to the
mark by absolute methanol to get stock solutions of 1.00 mg mL− 1. Into
100.00 mL volumetric flask, 1.00 mL of stock solutions was diluted by
the same solvent to get working solutions of 10.00 μg mL− 1. Stock and
working solutions of ATE, BIS, LAB NEB and PRO were prepared in the
same manner.
3.3. Pharmaceutical samples preparation
Twenty tablets of each Betaxolol 20 mg ® tablets and Carvid 25 mg ®
tablets were weighed and then grinded in a glass mortar. Amounts of the
powdered tablets equivalent to 2000.00 µg were transferred into 10.00
mL volumetric flasks followed by addition of 5.00 mL methanol (HPLC
grade), then sonicated for 30.00 min. The flasks were completed to the
mark and well shaken. Finally, contents were filtered through filter
paper, then one mL portions of the clear supernatants were transferred
into 10.00 mL volumetric flasks and completed to the mark by methanol
to get working solutions of 20.00 μg mL− 1.
3.4. Instrumentation
A UPLC (Dionex, Germany) instrument had been employed and was
equipped with a model series pump 3000, autosampler with variable
100.00 µL loop, thermostatted column compartment. A 50.00 × 2.10
mm (i.d.) Hypersil Gold C18 column (3.00 µm particle size) was used for
analysis. The instrument was supplied by a photodiode array detector
for quantification and the flow rate was kept at 1.00 mL/min. Data
acquisition was performed on Chromeleon 7.2 software. Isocratic
elution has been employed using different ratios of acetonitrile and
phosphate buffer at different buffer pH values as well as different col­
umn temperatures with DAD detection at 220 nm. The column was
equilibrated with mobile phase for saturation of the stationary phase
3
Journal of Chromatography B 1166 (2021) 122549
prior to chromatographic analysis. Ultrasonic cleaner (Cole-Parmer,
Chicago, USA), pH meter, model 3305 (Jenway, London, UK), Sartorius
handy balance–51 (Hanover, Germany) and rotatory oil vacuum pump
(Shimadzu, Kyoto, Japan) were used.
3.5. Method validation
Validation was carried out in accordance to ICH Q2 (R1) guidelines
[28] for the following parameters; LOD, LOQ, linearity, precision, ac­
curacy and robustness.
3.5.1. Limits of detection and quantitation (LOD and LOQ)
The LOD and LOQ are the least detected and the least quantified
limits, respectively. They were determined by Equations. (1) and (2),
respectively.
LOD = 3.3 × δ/S (1)
LOQ = 10 × δ/S (2)
where, δ is the standard deviation of the intercept and S is the slope of the
linearity plot
3.5.2. Linearity
Calibration lines were constructed by plotting peak areas versus
concentrations for serial dilutions of the investigated drugs were
analyzed in ranges; 5.00 – 50.00 and 5.00 – 55.00 μg mL− 1 for BEX and
CAR, respectively.
3.5.3. Accuracy
For proving the accuracy of the assay, a recovery study was carried
out. Standard solutions of investigated drugs of 20.00 μg/mL concen­
tration were spiked with accurate concentrations, corresponding to
approximately 80.00%, 100.00%, and 120.00% of the label claim (i.e. to
get total concentrations of 36.00 ,40.00 and 44.00 μg mL− 1). The sam­
ples were prepared in triplicates and analyzed in accordance with the
optimized analytical procedure. Further, mean recoveries, standard
deviation (SD) and percentage relative standard deviation (% RSD) were
calculated [29].
3.5.4. Precision
The precision of an analytical procedure is the closeness of agree­
ment or the degree of scatter between a series of measurements obtained
from multiple samples of the same homogeneous sample under pre­
scribed conditions. [30]. It is investigated at three concentrations within
linearity range for each drug (10.00, 20.00 and 40.00 μg/mL) in two
levels; intra-day precision or repeatability at different time intervals in
the same day and inter-day or intermediate precision as repetitions on
subsequent days. The values of mean recoveries, SD and % RSD were
also calculated.
3.5.5. Robustness
The robustness of an analytical procedure is a measure of its capacity
to remain unaffected by small, but deliberate variations in method pa­
rameters and provides an indication of its reliability of the developed
method. It was conducted on 10.00 μg/mL working standards of each
drug through investigation of the CMPs after faint alteration in the
studied CQAs like temperature by ± 2.00 ◦ C, pH of mobile phase by ±
0.10 and % acetonitrile by ± 0.20% v/v). Evaluation was carried out by
calculation of the % RSD values.
3.6. Design of experiments (DoE)
3.6.1. Selection of responses and experimental factors
The most effective chromatographic parameters which are consid­
ered as responses (CMPs) were selected, namely, peak area (A), peak
A.F.A. Hakiem et al. Journal of Chromatography B 1166 (2021) 122549

Table 1 where, Y is the predicted response; Xi and Xj are the independent vari­
The Box-Behnken design matrix with coded and original values for optimization
ables; β0 is the intercept (regression coefficient of the model); βi, βii, and
of the chromatographic method.
βij are the linear, quadratic and interaction coefficients, respectively.
Standard order Factors The model coefficients with statistical significance < 0.05 were
Temperature pH % Organic modifier
considered in framing the polynomial equation.
1 25.00 (− 1) 3.15 (− 1) 50.00 (0)
2 35.00 (+1) 3.15 (− 1) 50.00 (0)
3 25.00 (− 1) 4.55 (+1) 50.00 (0) 3.7. QSPR computational modelling
4 35.00 (+1) 4.55 (+1) 50.00 (0)
5 25.00 (− 1) 3.85 (0) 30.00 (− 1)
6 35.00 (+1) 3.85 (0) 30.00 (− 1) Based on partial least square (PLS) and multiple linear regression
7 25.00 (− 1) 3.85 (0) 70.00 (+1) (MLR) techniques, the (MOE 2019.01 software) [35] was utilized in
8 35.00 (+1) 3.85 (0) 70.00 (+1) order to find the appropriate statistical and mathematical models for
9 30.00 (0) 3.15 (− 1) 30.00 (− 1) prediction of retention. By involving additional structurally related beta-
10 30.00 (0) 4.55 (+1) 30.00 (− 1)
blockers, an accurate QSPR prediction was obtained using multivariate
11 30.00 (0) 3.15 (− 1) 70.00 (+1)
12 30.00 (0) 4.55 (+1) 70.00 (+1) methods to model relevant properties as a function of chromatographic
13 30.00 (0) 3.85 (0) 50.00 (0) behavior. The analysis was conducted on 315 molecular descriptors.
Using the contingency analysis [36] approach through the software, two
molecular descriptors provided the best correlated polynomial models.
width at 50% (W50%), asymmetry factor (T) and number of theoretical
Validation has been developed by cross validation (CV) that evaluates
plates (N) in addition to retention time (Rt), peak height (H) and ca­ how large the model applicability for a given data set. It has been carried
pacity factor (K’). The column temperature, mobile phase pH and per­ out by repeated regressions on data subsets by excluding each molecule
centage of organic modifier were selected to be optimized as three once (leave one out) and sometimes by more than one molecule (leave
critically or significantly affecting experimental parameters (CQAs) on many out, LMO), in turn, the correlation is measured using the predicted
the method CMPs. The column temperature corresponds to the low and values of the missing molecule/s. The developed final model of pre­
the high values affecting the viscosity of the eluent [31] while, pH value dicted statistics calculates the correlation coefficient of multiple deter­
of mobile phase corresponds to the low and the high pH values lower mination (R2), Equation (5)
than the smallest pKa and close to it [32]. Finally, the mobile phase ratio /
corresponds to the dependence of solute retention (k’) in LC on mobile ∑ ∑
phase composition represented in lower and higher values of acetoni­ R2 = 1 − (yei − yci )2 (yei − 〈yci 〉)2 (5)
i i
trile, this could be explored using Eq. (3)
log k’ = log kw − Sφ (3) where, i, the summation index and also the index of the i-th sample; ye,
experimental values of y and yc, the calculated values of y, i.e., values
where, kw, is the value of k’ with pure water as the mobile phase, φ is the from calibration.
volume fraction of the organic modifier, and S is a constant of a given In addition to the CV, the Root-mean squared error (RMSE) has been
solute and a given RP-HPLC system. The Box-Behnken design matrix was determined as an important and complementary validation parameter,
selected as a class of rotatable or nearly rotatable second-order designs Equation (6), which represents error between the mean of the experi­
based on three factors each with three levels usually afford reasonable fit mental values and predicted ones that was achieved for fitting of the
to experimental data within limited range of organic modifier percent­ developed QSPR models, and hence, explore the reliability of models if
ages [33]. The design matrix has enlisted the investigated factors and they possess predictive quality reflected on numerical values of corre­
their respective three equidistant levels were encoded as; the low (− 1), lation coefficients.
the intermediate (0) and the high (+1) levels, and it consisted of 17 ⎡ ( )2 ⎤
experiments with 5 replicates at the center point (13th experiment) to ⎢∑n ̂y i − ym ⎥
estimate the experimental error. Table 1 ⎢
RMSE = Sqrt⎢ i=1
⎥
⎥ (6)
⎣ n− 1 ⎦
The mutual interaction among the variables and their corresponding
optimum levels was expressed by a second order polynomial equation. A
generalized form is given in Equation (4) [34]
where, y and ŷ are the experimental and predicted RM values for an in­
∑ ∑ ∑
Y = β◦ + β i Xi + βii Xi2 + βij Xi Xj (4) dividual compound in the training set, ym is the mean of the experi­
mental RM values, and n is the number of molecules in the set of data

Table 2
Analysis of variance (ANOVA) for response surface quadratic model of chromatographic analysis of BEX.
Rt A H Width (50%) K’ T N
(inverse)* (Log10)*
CE p-value CE p-value CE p-value CE p-value CE p-value CE p-value CE p-value

Temperature (A) − 0.22 0.0628 0.001 0.6627 3.00 0.1964 − 0.08 0.0360 − 0.02 0.1939 0.69 0.0030 0.03 0.7540
pH of mobile phase (B) 0.08 0.4363 0.007 0.0598 − 8.94 0.0038 0.09 0.0196 0.10 0.0001 − 0.55 0.0098 − 0.32 0.0182
% Organic modifier (C) 0.22 0.0648 − 0.015 0.0011 4.78 0.0572 − 0.21 0.0002 0.01 0.5691 0.62 0.0056 0.81 0.0001
AB 0.05 0.7377 0.002 0.5971 − 1.48 0.6344 0.01 0.9078 0.02 0.2782 − 0.47 0.0733 − 0.06 0.7122
AC − 0.35 0.0433 0.0004 0.9134 2.69 0.3964 − 0.02 0.6323 0.06 0.0388 0.75 0.0119 − 0.11 0.4836
BC 0.01 0.9330 − 0.005 0.2811 7.66 0.0366 − 0.08 0.0881 0.01 0.5729 − 0.04 0.8529 0.00 0.9937
A2 0.12 0.4171 − 0.019 0.0023 4.98 0.1294 0.17 0.0045 − 0.08 0.0047 0.36 0.1396 − 0.33 0.0547
B2 − 0.14 0.3547 − 0.016 0.0058 10.67 0.0078 − 0.03 0.5126 0.03 0.1887 − 0.02 0.9153 0.21 0.1917
C2 0.16 0.2817 − 0.013 0.0128 7.66 0.0332 − 0.19 0.0020 − 0.01 0.7847 0.13 0.5760 0.93 0.0003
Intercept 2.15 0.1596 0.072 0.0032 20.71 0.0107 0.52 0.0016 1.15 0.0030 2.03 0.0076 1.98 0.0013
R2 0.74 0.93 0.89 0.94 0.95 0.90 0.94
Adequate precision 6.63 7.66 7.28 11.57 12.82 10.50 12.05
C.V. % 12.95 16.53 18.77 16.99 2.98 19.66 12.58

4
A.F.A. Hakiem et al. Journal of Chromatography B 1166 (2021) 122549

Table 3
Analysis of variance (ANOVA) for response surface quadratic model of chromatographic analysis of CAR.
Rt A H Width (50%) K’ T N
(inverse)* (Log10)*
CE p-value CE p-value CE p-value CE p- CE p-value CE p-value CE p-
value value

Temperature (A) 0.013 0.014 − 3.99 0.0096 − 0.07 0.0119 0.005 0.93 − 0.018 0.041 0.33 0.0024 0.002 0.85
pH of mobile − 0.005 0.27 − 3.74 0.0130 − 0.20 < 0.072 0.22 0.012 0.13 − 0.94 < 0.023 0.063
phase (B) 0.0001 0.0001
% Organic − 0.078 < 5.72 0.0015 0.01 0.5929 − 0.065 0.27 0.134 < − 0.14 0.0911 − 0.045 0.005
modifier (C) 0.0001 0.0001
AB − 0.016 0.031 − 2.85 0.1181 0.03 0.3393 0.005 0.95 0.023 0.051 − 0.80 < 0.003 0.85
0.0001
AC 0.067 < 14.00 < 0.10 0.0109 0.018 0.82 − 0.131 < 1.48 < 0.001 0.95
0.0001 0.0001 0.0001 0.0001
BC − 0.051 < − 1.60 0.3512 0.08 0.0273 − 0.273 0.01 0.094 < 0.13 0.2477 − 0.022 0.18
0.0001 0.0001
A2 0.011 0.085 10.08 0.0003 0.15 0.0015 0.029 0.71 − 0.015 0.175 0.69 0.0002 0.014 0.37
B2 0.011 0.096 14.25 < 0.25 < − 0.031 0.70 − 0.028 0.022 0.29 0.0211 − 0.021 0.20
0.0001 0.0001
C2 − 0.008 0.190 13.09 < 0.13 0.0025 − 0.070 0.40 0.023 0.0460 0.20 0.0861 − 0.041 0.0317
0.0001
Intercept 0.463 < 19.05 < 1.39 0.0001 0.603 0.22 0.065 < 1.78 < 0.117 0.048
0.0001 0.0001 0.0001 0.0001
R2 0.99 0.98 0.97 0.79 0.99 0.99 0.86
Adequate 32.70 16.08 15.23 5.74 34.76 26.56 5.93
precision
C.V. % 2.46 8.72 3.65 26.02 35.48 8.65 31.27

being examined [37,38].
3.8. Data analysis and optimization
The optimization of QBD data analysis was performed using Design
Expert® ver. 7.0.1 software (M/s Stat-Ease Inc., MN, USA) by multiple
linear regression analysis for estimating effects and interacting effects
through fitting the second-order quadratic polynomial model to the
experimental data. The model suitability was finally approved through
analysis of different parameters like coefficient of estimates (CE), the
associated probability values (p-value), correlation coefficient (R2),
percentage coefficient of variation (% CV) and precision. The 3D-
response surface plots were exploited for response surface analysis to
evaluate responses to each studied factor as well as interactions between
them. Numerical optimization was carried out on different CMPs to get
the acceptance criteria, like maximizing A, H and N in addition to
decreasing W50 % and T. After that, graphical optimization had been
done in order to get the analytical design space and, hence locating
optimum parameters.
4. Results and discussion
4.1. The influence of process variables
The 3-D surface plots in addition to the obtained p-values of ANOVA
tables, Tables 2 and 3 were utilized efficiently for checking to what
extent the effects of main factors (CQAs) and their binary interactions on
CMPs are significant. Close inspection of ANOVA tables has revealed
that temperature (factor A) exhibited significant effects on W50%, H and
T in case of BEX (p < 0.05). In contrast, temperature showed significant
effects on the overall CMPs except W50% and N in case of CAR.
Generally, T was the most affected CMP by temperature for both BEX
and CAR (p = 0.003 for BEX and 0.0024 for CAR). It is clearly observed
that factor B (pH of the mobile phase) has different effects on CMPs in
comparison to factor A. For BEX peaks, pH showed significant effects on
the whole investigated system suitability parameters except Rt and A
with p-values varied from 0.0196 in case of H down to 0.0001 for K’.
Among the investigated CQAs with CAR, it was noticed that pH has
moderate effect on A (p-value = 0.013) and strong significant ones on H
and T (p-values<0.0001). On the other hand, factor C (the percentage of
acetonitrile as an organic modifier), exhibited significant effects only on
A and N in case of BEX, with p-values of 0.0011 and 0.0001, respec­
tively. On the contrary, the whole CMPs were significantly influenced by
factor C in case of CAR with p-values varying from 0.005 with N down
to<0.0001 for both K’ and Rt. Exploring the obtained coefficient of es­
timate (CE) values have verified whether relations between CQAs and
CMPs are direct or inverse in accordance to the signs of their values. The
binary interactions between the studied CQAs and their effects on
selected CMPs have been clearly depicted by the 3D response surface
plots and the significant results were indicated by ANOVA analysis. With
respect to BEX peaks, it was observed that interaction between tem­
perature and pH of the mobile phase has no significant mutual effect on
any of the CMPs (p-values greater than 0.05, Table 2). In contrast, the
interaction between these factors showed significant inverse effects on
Rt and T of CAR (p < 0.05, Table 3). It was found that interaction be­
tween temperature and % organic modifier had exhibited significant
inverse effect on Rt and direct ones on both K’ and T of BEX (p < 0.05,
Table 2, Fig. 2). Similarly, interactive effects between temperature and
% organic modifier has significantly increased Rt and H of CAR, but
decreased its K’ and A (Table 3, Fig. 3). On the other hand, positive
interaction between mobile phase pH and % organic modifier was
evident for BEX in terms of its H values (Table 2). Concerning CAR, Rt
and W50% seemed to be significantly decreased, while H and K’ were
increased by increasing both temperature and pH of the mobile phase.
(Table 3, Fig. 3).
Determination of the optimum process values was conducted using
the Derringer’s desirability function method. Derringer’s desirability
function is a multi-criteria approach first introduced by Bourguignon
and Massar to determine chromatographic conditions that will result in
the most desirable combination of separation, sensitivity and analysis
time and hence the best chromatogram out of a number of possible
chromatograms [39]. It is based on the transformation of the measured
properties to a dimensionless desirability scale for each criterion, so that
values of several properties, obtained from different scales of measure­
ments, may be combined. The values for desirability ranges from zero
for the undesirable level of quality to the value of unity which indicates
an ultimate level of quality beyond which further improvements would
have no value [40]. This desirability function has the advantage that if
one of the criteria has an unacceptable value, then the overall product
will also be unacceptable. While, with the utility functions, this is not the

5
A.F.A. Hakiem et al.
Fig. 2. 3D-response surface plots of BEX chromatographic peaks showing the strong and moderate significant interaction effects between the studied CQAs, i.e.,
Temperature (A), mobile phase pH (B) and % organic modifier (C) on the investigated CMPs as represented in a, e and f as well as a strong significant effect of B on A
in b and C on H, W50% and N as represented in c. d and g, respectively.
case [41]. The main targets of the optimization were to maximize A, H
and N and recalculating all responsible factors by using desirability
functions, therefore, in the numerical optimization, the goal for column
temperature, pH of mobile phase, % organic modifier, K’ and Rt were
assigned as in range, while T was set to be minimized and N to be
maximized for both drugs. In order to improve desirability, getting much
less T and much higher N, while W50% was left as in range and mini­
mized for BEX and CAR, respectively. A weight factor of 3 (equals
importance) was chosen for all individual desirabilities in this work. By
applying the methodologies of the desired functions, the investigated
drugs have shown different ramp desirability values that was developed
from optimum points via numerical optimization of the studied CMPs
and these values are considered acceptable (the largest possible N as it is
an indication for column and separation efficiencies [42], T below 2.00
[43] and the largest possible A for improved sensitivity). Betaxolol HCl
has exhibited an excellent desirability of 0.82 for a temperature of
26.22 ◦ C, mobile phase pH of 4.55 and % organic modifier of 70.00.
While CAR has shown also an excellent desirability of 0.85 for the op­
timum parameters; temperature of 27.50 ◦ C, mobile phase pH of 4.55
and % organic modifier of 70.00. The optimum operational conditions of
the investigated drugs were defined at the high values of A, N as well as
low value of T as desired chromatographic system suitability parameters
required for efficient resolution, Figs. 4 and 5. The optimum
6
Journal of Chromatography B 1166 (2021) 122549
chromatograms have been illustrated Fig. 6.
4.2. Chemometric QSRR data analysis
The molecular descriptors are considered as independent variables,
while retention time as dependent one in a partial least square regres­
sion analysis using MOE as it can analyze noisy, collinear and incom­
plete variables in both X and Y matrices. It also provides large predictive
ability enabling establishing reliable QSPR models. Linear polynomial
multivariate models have been developed as well as simple linear
regression models has been established between the experimental Rt and
the predicted one. The developed models have exhibited good correla­
tion coefficients, Table 6 [44]. Standardization should be carried out
prior to analysis to avoid the effect of different scales in lipophilicity
evaluation. The analyzed data were scaled and mean-centered to unit
variance [45,46]. The descriptors were recalculated (normalized) by Z-
score scaling algorithm (V = mean of V/δ, where V is the value of var­
iables and δ is the standard deviation) then scaled. For feature de­
scriptors, if Z-score value is zero, this indicates that the feature is absent
[47,48]. In the current study, all the calculated values are larger than
zero and ranged from 0.14 to 1.65, not exceeding 2.50 proving that there
are no outliers [49], furthermore minimal residuals (RES) obtained
(difference between predicted value and experimental ones), Table 4
A.F.A. Hakiem et al.
Fig. 3. 3D-response surface plots of CAR chromatographic peaks showing the mild and moderate significant interaction effects between the studied CQAs, i.e.,
Temperature (A), mobile phase pH (B) and % organic modifier (C) on the investigated CMPs as represented a to j as well as a strong significant effect of C on N as
represented in k.
indicating that all investigated descriptors are featured, Table 7.
A number of polynomial models have been developed describing
relations of Rt values of investigated drugs and two molecular de­
scriptors: The octanol-water partition coefficient, logP(o/w) and the
electrostatic energy, E_ele. The logP(o/w) is kept in all developed
multivariate correlations as a basic parameter for lipophilicity mea­
surement because of its importance as the main determinant for drugs
retentions on different stationary phases [50]. The electrostatic energy is
considered as quantum-chemical descriptor while logP(o/w) is an
experimentally derived one [51]. The obtained numerical values of the
two descriptors are illustrated in Table 5.
The obtained R2 values at different ACN percentages were above
0.60, indicating the good correlations of the developed CV models of the
two descriptors [52,53]. The R2 values was found to be from 0.8765 to
0.9266, Table 4, indicating reliable regression statistics. The relatively
small values of the root mean square error (RMSE) as an important in­
ternal validation approach is is ranged from 0.68 to 1.10 indicating
reliability, Table 6.
7
Journal of Chromatography B 1166 (2021) 122549
The three-dimensional plots at different ACN percentages has shown
variations in correlations of the investigated drugs with respect to the
selected descriptors. Clustering is clearly observed between BET, NEB,
CAR, BIS and PRO with eluent systems of 64.00 and 67.00% ACN. This
could be attributed to relatively the same polarities, since, BEX, NEB and
BIS salts existed in their unionized forms at this acidic medium and their
polarities showed quiet different positioning owing to polarities of car­
bazol and naphthalene moieties corresponding to CAR and PRO,
respectively. Upon increasing ACN ratio to 70.00%, distortion of po­
larities of BEX, NEB and BIS salts might be due to varying ionization
status of these analytes, but at the same time CAR and PRO remained
unaffected. The modifications in polarity with respect to eluent system
accompanying ascending percentage of ACN as well as the imparted
polarities of analytes due to ionization could cause clustering of NEB and
BEX in addition to NEB and BIS with ACN percentages of 73.00 and
76.00%, respectively, Fig. 7.
By plotting the respective experimental Rt values against the calcu­
lated ones, good linear regression correlations have been obtained with
A.F.A. Hakiem et al.
Fig. 4. Desirability ramp plot for optimization of BEX.
R2 varied from 0.8765 to 0.9266, Table 6, hence the prediction capa­
bilities of the developed models are considered reliable.
4.3. Method validation studies
4.3.1. Linearity, LOD and LOQ
Linear calibration plots have been obtained within the studied con­
centration ranges of BEX and CAR with R2 values of 0.9985 and 0.9798,
respectively. The investigated drugs have shown LOD values of 1.80 and
2.00 as well as LOQ values of 5.52 and 6.31 µg/mL for BEX and CAR,
respectively, indicating high sensitivity.
4.3.2. Accuracy and precision
The intra- and inter-day precision studies have been conducted on
authentic samples of the studied drugs. Concerning the accuray study,
peak areas of tablet extracts were measured alone and then measured
after addition of 2.50 mL volume of three different concentrations of
standard solutions into fixed volumes (2.50 mL) of extract solutions. The
concentrations are then measured according to the following equation,
Eq. (7)
Cx / Cx + Cs = Ax / Ax+s (7)
where, Cx and (Cx + Cs) are the concentration of the analyte without and
with the standard addition, respectively, Ax and Ax+s are the signal in­
tensities of the solutions containing Cx and (Cx + Cs).
8
Journal of Chromatography B 1166 (2021) 122549
In accordance to Tables 8 and 9, good recovery percentages were
obtained ranged from 91.20 to 103.50 as well as percentage relative
standard deviation values not more than 2.50% indicating that the
developed method represents reliable analysis of BEX and CAR in quality
control laboratories.
4.3.3. Robustness
Faint changes have been applied to the optimized chromatographic
conditions (CQAs) and the extent of change in selected CMPs (i.e. A, T,
W50% and N) were represented as %RSD values as an indication of the
method robustness. The % RSD values ranged from 0.006 up to 2.00%
resumes the robustness of the proposed method. The results were sum­
marized in Table 10.
5. Conclusion
The present study has demonstrated successful utilization of QBD for
development of robust HPLC analysis of two representative β.blockers,
namely; BEX and CAR using Box-Behnken design. The effect of three
selected CQAs on a number of CMPs was investigated. A reliable
determination of BEX and CAR was obtained at column temperatures of
26.00 and 27.50 ◦ C, respectively using acetonitrile and phosphate buffer
(pH 4.55) 70:30 v/v at flow rate 1.00 mL/min and detection wavelength
of 220 nm. The desirability function was exploited as a tool for setting
the optimized chromatographic conditions as represented in the devel­
oped analytical design spaces with excellent values of 0.82 and 0.85 for
A.F.A. Hakiem et al. Journal of Chromatography B 1166 (2021) 122549

Fig. 5. Desirability ramp plot for optimization of CAR.

Fig. 6. Optimized chromatographic bands of the investigated drugs.

BEX and CAR, respectively. Experimental validation study has been
conducted owing to ICH guidelines and has revealed acceptable values.
A complementary and confirmatory QSPR study has been conducted to
seven β.blockers (BET, CAR and five other β.blockers) using retention
time as target property at variable ACN ratios for detecting the best
correlated molecular descriptors to the retention behavior. Upon
development of QSPR models coupled with multiple linear regression
analysis, it was found that retention of analytes is greatly dependent on
the lipophilicity (logP(o/w) and the E_ele as common descriptors with
good correlation coefficients ranged from 0.8765 to 0.9266. The high
performance of validation has been greatly affected by the developed
models. The developed QSPR study exploiting the conventional features

9
A.F.A. Hakiem et al. Journal of Chromatography B 1166 (2021) 122549

Table 4 Table 6
Input normalization and scaling. Two descriptor polynomial linear regression models
Eluent system Analyte Rt (EXP) Rt RESb Z- Eluent systems Estimated linear models R2 Root mean square
(PRED)a SCOREc error (RMSE)*

Phosphate buffer ATE 2.7500 3.0100 − 0.2596 0.2372 Phosphate buffer Rt = -21.852 + 9.724 logP 0.8765 1.0941
pH 4.55 – ACN BIS 7.8000 5.8817 1.9182 1.7531 pH 4.55 – ACN (o/w) − 0.863 E_ele (6)
24: 76, v/v BEX 2.6700 3.9858 − 1.3158 1.2026 24: 76, v/v
CAR 2.2400 1.8619 0.3780 0.3454 Phosphate buffer Rt = 20.436 + 9.208 logP 0.8899 0.9496
LAB 10.5000 11.3093 − 0.8093 0.7396 pH 4.55 – ACN (o/w) − 0.803 E_ele (7)
NEB 2.2300 3.2104 − 0.9804 0.8960 27: 73, v/v -
PRO 7.0000 5.9309 1.0690 0.9770 Phosphate buffer Rt = -20.5914 + 9.176 logP 0.9113 0.8379
Phosphate buffer ATE 2.6800 2.8158 − 0.1358 0.1430 pH 4.55 – ACN (o/w) − 0.799 E_ele (8)
pH 4.55 – ACN BIS 7.3000 5.7322 1.5677 1.6507 30: 70, v/v
27: 73, v/v BEX 2.6600 4.0370 − 1.3770 1.4500 Phosphate buffer Rt = 18.7259 + 8.407 logP 0.8912 0.8598
CAR 2.6600 2.2636 0.3963 0.4173 pH 4.55 – ACN (o/w) − 0.732 E_ele (9)
LAB 10.2000 10.8310 − 0.6310 0.6642 33: 67, v/v
NEB 2.6600 3.3976 − 0.7376 0.7767 Phosphate buffer Rt = -18.3922 + 8.209 logP 0.9266 0.6780
PRO 6.8000 5.8826 0.9173 0.9660 pH 4.55 – ACN (o/w) − 0.717 E_ele (10)
Phosphate buffer ATE 2.5299 2.5499 − 0.0199 0.0238 36: 64, v/v
pH 4.55 – ACN BIS 6.5000 5.4755 1.0244 1.2226
30: 70, v/v BEX 2.5799 3.7961 − 1.2161 1.4513
CAR 2.4200 2.0525 0.3675 0.4385
LAB 10.0000 10.5519 − 0.5519 0.6586 Table 7
NEB 2.4000 3.1683 − 0.7683 0.9169 Correlation analysis between predicted retention times and experimental ones at
PRO 6.8000 5.6355 1.1644 1.3896 different ACN percentages .
Phosphate buffer ATE 2.5000 2.4757 0.0242 0.0282
pH 4.55 – ACN BIS 6.1999 5.1559 1.0440 1.2142 Eluent systems Estimated linear models R2
33: 67, v/v BEX 2.2699 3.6172 − 1.3472 1.5668 Phosphate buffer pH 4.55 – Rt (Pred.) = 0.8765 Rt (Exp.) + 0.621 0.8765
CAR 2.4800 2.0197 0.4602 0.5353 ACN (11)
LAB 9.3000 9.8068 − 0.5068 0.5894 24: 76, v/v
NEB 2.2699 3.0420 − 0.7720 0.8979 Phosphate buffer pH 4.55 – Rt (Pred.) = 0.89 Rt (Exp.) + 0.55 (12) 0.8899
PRO 6.4000 5.3024 1.0975 1.2764
ACN -
Phosphate buffer ATE 2.4600 2.3488 0.1112 0.1639
27: 73, v/v
pH 4.55 – ACN BIS 5.3000 4.9408 0.3591 0.5297 Phosphate buffer pH 4.55 – Rt (Pred.) = 0.9113 Rt (Exp.) + 0.4210 0.9113
36: 64, v/v BEX 2.3800 3.4254 − 1.0454 1.5419 ACN (13)
CAR 2.0799 1.8348 0.2451 0.3615 30: 70, v/v
LAB 9.1000 9.4877 − 0.3877 0.5719 Phosphate buffer pH 4.55 – Rt (Pred.) = 0.8912 Rt (Exp.) + 0.4884 0.8912
NEB 2.3399 2.8516 − 0.5116 0.75456 ACN (14)
PRO 6.3000 5.0706 1.2293 1.8131 33: 67, v/v
a
Predicted value calculated by MOE software. Phosphate buffer pH 4.55 – Rt (Pred.) = 0.9266 Rt (Exp.) + 0.3138 0.9267
b ACN (15)
Difference between the value of the model (predicted value) and the activity
36: 64, v/v
field (experimental value).
c
Absolute difference between the value of the model calculated by MOE
software and the activity field (Experimental value), divided by the square root Declaration of Competing Interest
of the mean square error of the dataset.

The authors declared that there is no conflict of interest.

Table 5
References
Numerical values of the best correlated molecular descriptors; with respect to
the studied analytes.
[1] Requirements for Registration of Pharmaceuticals for Human Use - Guildlines for
Analytes Molecular Descriptors Elemental Impurities, ICH Harmon. Guidel. Pharm. Dev. Q8(R2). (2009).
logP(o/w) E_ele [2] K. Nidhi, S. Indrajeet, M. Khushboo, K. Gauri, D.J. Sen, Development of Quality-By-
Design Analytical Methods, J. Pharm. Sci. 100 (2011) 797–812.
ATE 0.7319 − 20.5503 [3] P.F. Gavin, B.A. Olsen, A quality by design approach to impurity method
BIS 2.2637 − 6.6244 development for atomoxetine hydrochloride (LY139603), J. Pharm. Biomed. Anal.
BET 2.6979 0.4626 46 (2008) 431–441.
CAR 3.9747 17.3030 [4] David J. AM Ende, Chemical Engineering in the Pharmaceutical Industry, 1st ed.,
LAB 2.5639 − 9.5286 John Wiley & Sons, Inc., Hoboken, New Jersey, 2011.
NEB 3.2149 7.1840 [5] R. Peraman, K. Bhadraya, Y. Padmanabha Reddy, Analytical quality by design: A
PRO 2.9210 0.7215 tool for regulatory flexibility and robust analytics, Int. J. Anal. Chem. (2015),
https://doi.org/10.1155/2015/868727.
[6] A.S. Rathore, Quality by Design (QbD) -Based Process Development for Purification
of a Biotherapeutic, Trends Biotechnol. 34 (2016) 358–370.
selection algorithm in order to generate robust predictive comparable [7] T. Ns, A. Sv, Implementing Quality by Design (QbD) in Chromatography, Austin J.
models that could have generalized analytical application for the whole Anal. Pharm. Chem. 4 (2017) 1–5.
group members together with the optimized QBD models as comple­ [8] T. Baczek, R. Kaliszan, K. Novotná, P. Jandera, Comparative characteristics of
HPLC columns based on quantitative structure-retention relationships (QSRR) and
mentary approaches and hence reduce the required time and cost. hydrophobic-subtraction model, J. Chromatogr. A 1075 (2005) 109–115.
[9] M. Kaczmarek, A. Buciński, M.P. Marszałł, A. Badura, R. Kaliszan, Thermodynamic
CRediT authorship contribution statement and QSRR modeling of HPLC retention on modern stationary phases, J. Liq.
Chromatogr. Relat. Technol. 38 (2015) 62–67.
[10] M.A. Fouad, E.H. Tolba, M.A. El-Shal, A.M. El Kerdawy, QSRR modeling for the
Ahmed Faried Abdel Hakiem: Methodology, Investigation, Soft­ chromatographic retention behavior of some β-lactam antibiotics using forward
ware, Validation, Supervision. Ahmed K. Hamdy: Methodology, and firefly variable selection algorithms coupled with multiple linear regression,
J. Chromatogr. A 1549 (2018) 51–62.
Investigation, Data curation. Hassan Refat Hassan Ali: . Mohamed
[11] J. Ghasemi, S. Saaidpour, QSRR Prediction of the Chromatographic Retention
Gomaa: Software, Investigation, Data curation. Ahmed Safwat Abor­ Behavior of Painkiller Drugs, J. Chromatogr. Sci. 47 (2009) 156–163.
aia: Software, Investigation, Validation, Data curation.

10
A.F.A. Hakiem et al. Journal of Chromatography B 1166 (2021) 122549

Fig. 7. 3D plots for the two parameters correlation between Rt and the investigated molecular descriptors at different mobile phase systems.

Table 8
Accuracy study.
Analyte Equivalent concentration (µg/mL) of tablet Level % Total concentration (µg/mL) after standard Amount recovered (µg/mL) Recovery RSD
formulations addition ± SD % %

BEX 20.00 80.00 36.00 34.56 ± 1.24 96.00 3.60

100.00 40.00 39.30 ± 1.03 98.20 2.62
120.00 44.00 43.00 ± 0.56 97.55 1.30
CAR 20.00 80.00 36.00 35.66 ± 0.63 99.06 1.76
100.00 40.00 40.53 ± 1.60 101.32 0.40
120.00 44.00 43.30 ± 1.34 98.41 3.10

[14] N.S. Quiming, N.L. Denola, S.R. Bin Samsuri, Y. Saito, K. Jinno, Development of
Table 9 retention prediction models for adrenoreceptor agonists and antagonists on a
Intra- and inter-day precision study. polyvinyl alcohol-bonded stationary phase in hydrophilic interaction
chromatography, J. Sep. Sci. 31 (2008) 1537–1549.
Analyte Standard Amount recovered Recovery RSD [15] N.S. Quiming, N.L. Denola, I. Ueta, Y. Saito, S. Tatematsu, K. Jinno, Retention
concentration (µg/mL) (µg/mL) ± SD % % prediction of adrenoreceptor agonists and antagonists on a diol column in
hydrophilic interaction chromatography, Anal. Chim. Acta 598 (2007) 41–50.
BET Intra-day precision [16] H. Noorizadeh, M. Noorizadeh, A. Farmany, Advanced QSRR models of
10.00 9.45 ± 0.13 94.50 1.40 toxicological screening of basic drugs in whole blood by UPLC-TOF-MS, Med.
20.00 19.82 ± 0.45 99.12 2.30 Chem. Res. 21 (2012) 4357–4368.
40.00 40.93 ± 0.35 102.33 0.85 [17] B. Andri, A. Dispas, R.D.E.A. Marini, P. Hubert, P. Sassiat, R. Al Bakain,
Inter-day precision D. Thiébaut, J. Vial, Combination of partial least squares regression and design of
10.00 9.66 ± 0.24 96.64 2.50 experiments to model the retention of pharmaceutical compounds in supercritical
20.00 18.51 ± 0.22 92.51 1.20 fluid chromatography, J. Chromatogr. A 1491 (2017) 182–194, https://doi.org/
40.00 36.50 ± 0.81 91.20 2.22 10.1016/j.chroma.2017.02.030.
CAR Intra-day precision [18] R. Put, C. Perrin, F. Questier, D. Coomans, D.L. Massart, Y. Vander Heyden,
Classification and regression tree analysis for molecular descriptor selection and
10.00 9.32 ± 0.05 93.20 0.54
retention prediction in chromatographic quantitative structure-retention
20.00 20.30 ± 0.45 101.34 2.20
relationship studies, J. Chromatogr. A 988 (2003) 261–276.
40.00 41.40 ± 0.80 103.50 1.93
[19] B. Dejaegher, Y. Vander Heyden, HILIC methods in pharmaceutical analysis, J. Sep.
Inter-day precision Sci. 33 (2010) 698–715.
10.00 9.80 ± 0.21 98.05 2.14 [20] N.S. Quiming, N.L. Denola, Y. Saito, K. Jinno, Retention prediction of
20.00 20.02 ± 0.04 100.10 0.20 adrenoreceptor agonists and antagonists on unmodified silica phase in hydrophilic
40.00 39.30 ± 0.03 98.23 0.10 interaction chromatography, Anal. Bioanal. Chem. 388 (2007) 1693–1706.
[21] M. Goodarzi, R. Jensen, Y. Vander Heyden, QSRR modeling for diverse drugs using
different feature selection methods coupled with linear and nonlinear regressions,
[12] R. Kaliszan, Quantitative structure –(chromatographic) retention relationships, J. Chromatogr. B Anal. Technol. Biomed Life Sci. 910 (2012) 84–94.
Chem. Rev. 107 (2007) 3212–3246. [22] K. Jinno, N.S. Quiming, N.L. Denola, Y. Saito, Modeling of retention of
[13] J. Leszczynski, Challenges and Advances in Computational Chemistry and Physics, adrenoreceptor agonists and antagonists on polar stationary phases in hydrophilic
Springer Science+Business Media, Dordrecht Heidelberg London, 2019. interaction chromatography: A review, Anal. Bioanal. Chem. 393 (2009) 137–153.

11
A.F.A. Hakiem et al. Journal of Chromatography B 1166 (2021) 122549

Table 10
Robustness study.
Analyte Condition Variation Mean A (µA) ± SD RSD % Mean W 50% ± SD RSD % Mean N ± SD RSD % Mean T ± SD RSD %

BEX Temperature 24.22 569826.00 ± 3250.00 0.60 0.36 ± 0.0002 0.05 3153.00 ± 10.21 0.32 1.55 ± 0.0001 0.006
(oC) 28.22 569863.00 ± 427.00 0.07 0.33 ± 0.003 1.00 3168.12 ± 35.22 1.11 1.65 ± 0.01 0.60
pH 4.45 569882.00 ± 4536.00 0.80 0.28 ± 0.005 1.78 3242.05 ± 9.32 0.30 1.25 ± 0.004 0.32
4.65 569980.00 ± 4210.00 0.74 0.32 ± 0.003 0.93 3189.15 ± 15.30 0.50 1.60 ± 0.001 0.10
ACN % 69.80 568200.00 ± 3820.00 0.67 0.38 ± 0.01 1.50 3185.10 ± 11.24 0.35 1.15 ± 0.003 0.30
70.20 570721.00 ± 515.00 0.10 0.25 ± 0.002 0.80 3275.20 ± 25.00 0.76 0.96 ± 0.005 0.52
CAR Temperature 25.50 455233.00 ± 1520.00 0.33 0.30 ± 0.005 1.66 6865.03 ± 8.63 0.12 1.05 ± 0.02 2.00
(oC) 29.50 454350.00 ± 4100.00 1.00 0.31 ± 0.0004 0.13 6900.30 ± 18.24 0.30 1.12 ± 0.01 0.90
pH 4.45 454691.00 ± 860.00 0.20 0.25 ± 0.003 1.20 6814.25 ± 30.42 0.45 0.82 ± 0.005 0.61
4.65 455830.00 ± 2732.00 0.60 0.29 ± 0.005 1.66 6724.00 ± 19.33 0.30 1.13 ± 0.003 0.30
ACN % 69.80 454444.00 ± 3020.00 0.66 0.33 ± 0.0001 0.03 6785.40 ± 11.05 0.20 1.42 ± 0.02 1.41
70.20 455813.00 ± 1650.00 0.40 0.23 ± 0.003 1.30 6895.10 ± 15.12 0.23 0.90 ± 0.0001 1.11

*The values at the optimized conditions are 569100, 0.31, 3175 and 1.30for A, W50%, N and T of BEX , respectively, while those for CAR are 455621, 0.28, 6795 and
0.85, respectively.

[23] P. Wiczling, P. Kawczak, A. Nasal, R. Kaliszan, Simultaneous determination of pKa
and lipophilicity by gradient RP HPLC, Anal. Chem. 78 (2006) 239–249.
[24] T. Hamoir, B. Bourguignon, D.L. Massart, Validation of a strategic approach to
high-performance liquid chromatographic method selection, Chromatographia 39
(1994) 339–345.
[25] T. Baczek, R. Kaliszan, Quantitative structure/retention relationships in affinity
chromatography, J. Biochem. Bioph. Methods 49 (2001) 83–98.
[26] R. Kaliszan, Retention data from affinity high-performance liquid chromatography
in view of chemometrics, J. Chromatogr. B Biomed. Appl. 715 (1998) 229–244.
[27] K. Ciura, M. Belka, P. Kawczak, T. Bączek, M.J. Markuszewski, J. Nowakowska,
Combined computational-experimental approach to predict blood–brain barrier
(BBB) permeation based on “green” salting-out thin layer chromatography
supported by simple molecular descriptors, J. Pharm. Biomed. Anal. 143 (2017)
214–221.
[28] International conference on harmonization (ICH) Topic Q2 (R1): Validation of
analytical procedures: text and methodologyCurrent Step 4 version Parent
Guideline dated 27 October 1994 (Complementary Guideline on Methodology
dated 6 November 1996 incorporate, European Union, Japan and USA, 2005.
[29] J. Ermer, P. Nethercote, Method validation in pharmaceutical analysis A guide to
best practice, Second edi, WILEY-VCH Verlag GmbH & Co, KGaA, 2015.
[30] C.C. Chan, Y.C. Lee, H. Lam, X.-M. Zhang, Analytical method validation and
instrument performance verification, First edit, John Wiley & Sons Inc., Hoboken,
New Jersy, 2004.
[31] E. Katz, K. Ogan, R.P.W. Scott, Effect of pressure on solute diffusivity, solvent
viscosity and column temperature in liquid chromatography, J. Chromatogr. A 260
(1983) 277–295.
[32] K. Škrášková, L.H.M.L.M. Santos, D. Šatínský, A. Pena, M.C.B.S.M. Montenegro,
P. Solich, L. Nováková, Fast and sensitive UHPLC methods with fluorescence and
tandem mass spectrometry detection for the determination of tetracycline
antibiotics in surface waters, J. Chromatogr. B Anal. Technol. Biomed, Life Sci. 927
(2013) 201–208.
[33] V.J. Barwick, Strategies for solvent selection - A literature review, TrAC - Trends
Anal. Chem. 16 (1997) 293–309.
[34] K.M. Abdel-Gawad, A.F. Hifney, M.A. Fawzy, M. Gomaa, Technology optimization
of chitosan production from Aspergillus niger biomass and its functional activities,
Food Hydrocoll. 63 (2017) 593–601, https://doi.org/10.1016/j.
foodhyd.2016.10.001.
[35] Molecular Operating Environment (MOE), Chemical Computing Group Inc.,
Montreal, QC, Canada, 2018.
[36] R.V. Hogg, E.A. Tanis, D.L. Zimmerman, C. Town, Probability and Statistical
Inference, Ninth edit Pearson Education Inc., New Jersy, 2015.
[37] R. Veerasamy, H. Rajak, A. Jain, S. Sivadasan, C.P. Varghese, R.K. Agrawal,
Validation of QSAR Models - Strategies and Importance, Int. J. Drug Des. Discov. 2
(2011) 511–519.
[38] R. Kiralj, M.M.C. Ferreira, Basic Validation Procedures for Regression Models in
QSAR and QSPR Studies: Theory and Application, J. Brazilian Chem. Soc. 20
(2009) 770–787.
[39] M. Jimidar, B. Bourguignon, D.L. Massart, Application of Derringer’s desirability
function for the selection of optimum separation conditions in capillary zone
electrophoresis, J. Chromatogr. A 740 (1996) 109–117.
[40] F. Safa, M.R. Hadjmohammadi, Simultaneous optimization of the resolution and
analysis time in micellar liquid chromatography of phenyl thiohydantoin amino
acids using Derringer’s desirability function, J. Chromatogr. A 1078 (2005) 42–50.
[41] G. Derringer, R. Suich, Simultaneous Optimization of Several Response Variables,
J. Qual. Technol. 12 (1980) 214–219.
[42] A. de Villiers, F. Lestremau, R. Szucs, S. Gélébart, F. David, P. Sandra, Evaluation of
ultra performance liquid chromatography. Part I. Possibilities and limitations,
J. Chromatogr. A 1127 (2006) 60–69.
[43] L. Linda, Reviewer Guidance - Validation of chromatographic methods, CDER.
Cent. Drug Eval. Res. 22 (1998) 1–30. http://www.scopus.com/inward/record.url
?eid=2-s2.0-0031924265&partnerID=tZOtx3y1.
[44] S. Wold, M. Sjostrom, L. Eriksson, PLS-regression : a basic tool of chemometrics,
Chemom. Intell. Lab. Syst. 58 (2001) 109–130.
[45] K. Ciura, A. Rutecka, P. Kawczak, J. Nowakowska, Quantitative Structure –
Retention Relationship Modeling of the Retention Behavior of Selected
Antipsychotic Drugs, J. Planar Chromatogr. – Mod. TLC. 30 (2017) 225–230,
https://doi.org/10.1556/1006.2017.30.3.13.
[46] J. Nowakowska, K. Ciura, P. Kawczak, B. Wielgomas, T. Ba, Reversed-Phase and
Normal-Phase Thin-Layer Chromatography and Their Application to the
Lipophilicity Prediction of Synthetic Pyrethroids Based on Quantitative Structure-
Retention Relationships, J. Planar Chromatogr. – Mod. TLC. 31 (2018) 99–104.
[47] L.M. Hall, D.W. Hill, M. Chen, H. Lowell, D.F. Grant, Optimizing artificial neural
network models for metabolomics and systems biology : an example using HPLC
retention index data, Bioanalysis. 7 (2015) 939–955.
[48] P. Kawczak, K. Ewa, H. Kapica, J. Nowakowska, Application of reversed-phase thin
layer chromatography and QSRR modelling for prediction of protein binding of
selected β-blockers, J. Pharm. Biomed. Anal. 176 (2019), https://doi.org/10.1016/
j.jpba.2019.07.015.
[49] A.H. Rageh, N.N. Atia, H.M. Abdel-Rahman, Application of salting-out thin layer
chromatography in computational prediction of minimum inhibitory concentration
and blood-brain barrier penetration of some selected fluoroquinolones, J. Pharm.
Biomed. Anal. 159 (2018) 363–373.
[50] E.H.M.A. Rabtti, V.E. Vajs, RP TLC-Based Lipophilicity Assessment of Some Natural
and Synthetic Coumarins, J. Brazilian Chem. Soc. 23 (2012) 522–530.
[51] M. Dehmer, K. Varmuza, D. Bonchev, Statistical Modelling of Molecular
Descriptors in QSAR/QSPR, Second Edi, Wiley-Blackwelk, Vienna, 2012.
[52] A. Tropsha, P. Gramatica, V.K. Gombar, Validation is the Absolute Essential for
Successful Application and Interpretation of QSPR Models, QSAR Comb. Sci. 22
(2003) 69–77.
[53] A. Golbraikh, A. Tropsha, Beware of q 2 !, J. Mol. Graph. Model. 20 (2002)
269–276.

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