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Predictive retention study of β-blockers in

different chromatographic systems as potential
tool for lipophilicity screening based on QSRR
approach

Ahmed Faried Abdel Hakiem, Ahmed Mohsen Kamal, Al Montaser Bellah H.

Ali, Rofaida A. Salem & Ahmed Safwat Aboraia

To cite this article: Ahmed Faried Abdel Hakiem, Ahmed Mohsen Kamal, Al Montaser Bellah H.
Ali, Rofaida A. Salem & Ahmed Safwat Aboraia (2021) Predictive retention study of β-blockers
in different chromatographic systems as potential tool for lipophilicity screening based on QSRR
approach, Journal of Liquid Chromatography & Related Technologies, 44:1-2, 12-24, DOI:
10.1080/10826076.2020.1859384

To link to this article: https://doi.org/10.1080/10826076.2020.1859384

Published online: 16 Feb 2021.

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JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES
2021, VOL. 44, NOS. 1–2, 12–24
https://doi.org/10.1080/10826076.2020.1859384

Predictive retention study of b-blockers in different chromatographic systems as

potential tool for lipophilicity screening based on QSRR approach
Ahmed Faried Abdel Hakiema , Ahmed Mohsen Kamalb, Al Montaser Bellah H. Alic, Rofaida A. Salemd, and
Ahmed Safwat Aboraiab
a
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt; bMedicinal Chemistry
Department, Faculty of Pharmacy, Assiut University, Assiut, Egypt; cPharmaceutical Analytical Chemistry Department, Faculty of Pharmacy,
Assiut University, Assiut, Egypt; dPharmaceutical Chemistry Department, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt

ABSTRACT KEYWORDS
The chromatographic analysis of a number of b-blockers on different thin-layer chromatography b-Blockers; contingency
(TLC) systems was carried out. The analysis was done on three different systems, namely normal analysis; molecular
phase, paraffin oil impregnated in methylene chloride and n-hexane as two types of reversed descriptors; physicochemical
properties;QSRR
phase systems. This represents an economic and environmental approach pathway for the deter-
mination of four solvents systems of two components were used to get the retention parameters
and the pH was adjusted using ammonia and glacial acetic acid. Quantitative structure–retention
relationship (QSRR) was applied to the different phases and 315 molecular descriptors has been
calculated for each b-blocker. A contingency analysis has been used to declare the highest correl-
ation between the chromatographic retention and the calculated descriptors, which resulted in
obtaining different models for each system used. The produced models correlate the predicted
retention to the experimental one in a significant way which could be useful for the future behav-
ior prediction of this group of b-blockers against normal and reversed phase TLC systems.

GRAPHICAL ABSTRACT

Introduction different applicability in the management of cardiovascular

Along three decades and more, beta-blockers have been
widely used as first line for hypertension management.
National and international guidelines still recommend this
group as first-line although their suboptimal capability in
lowering blood pressure.[1]
Beta blockers are considered a heterogeneous group. As
they show variable receptor selectivity, intrinsic sympatho-
mimetic activity, vasodilating properties and metabolism and
hence differences in duration of action. Accordingly,
diseases owing to their physiopathological mechanisms.[2]
Since the great response diversity among this group, nine
commonly prescribed members were selected for this study.
As a common structural feature, all currently available beta
blockers possess an amino-alkanol side chain and an aro-
matic group: phenyl with atenolol (ATE), bisoprolol (BIS),
betaxolol (BET), carvidolol (CAR), labetalol (LAB), and sota-
lol (SOTA) as well as naphthyl, carbazol, chromen and thia-
diazole moieties in the case of propranolol (PRO), CAR,
nebivolol (NEB) and timolol (TIM), respectively.

CONTACT Ahmed Faried Abdel Hakiem midomycin500@yahoo.com Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Kafrelsheikh
University, Kafrelsheikh 71111, Egypt.
ß 2021 Taylor & Francis Group, LLC

Propranolol HCl is considered the most hydrophobic com-
pound among all beta blockers, owing to this fact, it has a
membrane stabilizing activity and nonselective beta recep-
tors antagonistic activity.[3]
Thin-layer chromatography (TLC) is considered a simple,
cheap and accurate analytical technique. Although TLC has
its own right, it can be utilized as a complimentary analyt-
ical tool to other chromatographic and spectroscopic techni-
ques and vice versa. Few adjustments in eluent composition
for separation of multi-component samples can be trans-
ferred from TLC to HPLC and fractions of interest can be
collected of HPLC system then re-chromatographed with
TLC giving fine tuned separation. This technique can be
hyphenated with many analytical systems to get far more
details about chromatographic separation in accordance to
chemical structures.[4] Additionally, TLC studies represent
one of the most important key identity tests in most
pharmacopeial monographs. These pharmacopeial standards
are utilized in industry as a basis of quality control (QC)
and current good manufacturing practices (cGMP)
requirements.[5]
Many researches discussed TLC analysis of beta blockers.
Up to 16 members beta receptors antagonists were resolved
efficiently by two-dimensional TLC and detected through
dansylation of the secondary amino group of the aliphatic
propranolamine side chain.[6] Systems of methanol either
with chloroform or ethylacetate in 25% ammonia were uti-
lized for efficient separation of ATN and PRO and in blood
plasma samples after liquid–liquid extraction with UV at
254 nm.[7,8] Nebivolol and CAR were analyzed on normal
phase plates either native or in tablet formulations.[9–11]
Dabsylation of BIS and LAB into visible light absorbing
derivatives was utilized for their densitometric analysis in
tablet formulations.[12] Stability indicating assay were carried
out for BET and NEB in their pharmaceutical formula-
tions.[13,14] Timolol, SOT, PRO and ATE extracted of urine
samples exhibited variable retentions and their chromato-
grams were subjected to four stages derivatization visible
detection.[15] Enantiomers of both ATE and PRO were
resolved using vancomycin as impregnating agent or as
Chiral Mobile phase additive.[16] Impregnated stationary
phases in bovine serum albumin were utilized for enantio-
meric separation of ATE, BIS, CAR and PRO.[17]
The experimental hydrophobicity or lipophilicity factors
are greatly correlated to the biological activity of the investi-
gated beta blockers. Many methods have been developed for
determination of logarithm of n-octanol–water partition
ratio (log Pow) but chromatographic methods are the most
common. This physicochemical property is considered the
main controller of chromatographic retention. Accordingly,
there is a great demand for improving accurate chromato-
graphic methods for fast prediction of biological activity,
which is especially important when high-throughput screen-
ing methods are used in drugs analysis. Generally, estima-
tion of such relationships is known as quantitative
structure–activity relationships (QSAR), or quantitative
structure–retention relationships (QSRR) from the chroma-
tographic point of view. Computational chemistry confirmed
JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 13
by experimental results introduces rapid and cheap means
in the pharmaceutical industry. However, calculated values
may vary with those observed in experiment. These signifi-
cant deviations could be attributed to nonbonded intramo-
lecular interactions, and calculated values do not match
those observed in experiment.[18]
The role of lipophilicity in drug–biomacromolecule inter-
actions has been extensively discussed in terms of quantita-
tive structure–property relationships (QSPR). Distribution of
pharmaceuticals into living system is represented as a series
of partitioning ways in conjunction with diffusion mecha-
nisms. Since the great relation of lipophilicity to molecular
size, hydrogen bonding and polarity of the molecules, it is
considered as one of the most important physicochemical
properties that determines the affinity of drugs to biological
membranes. hence, it manages pharmacokinetic behavior of
medicaments.[19] Different impregnation media for pre-
coated commercial silica gel plates were explored imparting
modified properties of normal phase like ion exchange, ion
pairing and lipophilicity in order to enhance resolution of
multi-component samples. Silica gel plates were selected for
its wide efficient use as its hydrated form contains free sila-
nol, geminal, and associated silanols.[20] This study investi-
gates significant differences in chromatographic retention of
the previously mentioned structurally related beta blockers
eluted by mobile phase system of different polarities and pH
values on silica gel plates of variable lipophilicities. The
obtained retention values of analytes were correlated to a
large number of molecular descriptors and hence by statis-
tical analysis, an accurate predictions for their chromato-
graphic behavior providing cost saving and tedious
optimization trials for future measurements.
RM ¼ log ð1=1  RF Þ (1)
where RF is the retardation factor and calculated as:
RF ¼ LS =Lm (2)
where Ls and Lm are distance traveled by analyte and solvent
front, respectively.[21]
Materials
The chemical structures of the investigated b-blockers are; -
2-[4-[2-hydroxy-3-(propan-2-ylamino) propoxy] phenyl]
acetamide for ATE, 1-(propan-2-ylamino)  3-[4-(2-propan-
2-yloxyethoxymethyl) phenoxy] propan  2-ol. for BIS, 1-[4-
[2-(cyclopropylmethoxy) ethyl] phenoxy]  3-(propan-2-yla-
mino) propan  2 – ol. HCl for BET, 1-(9H – carbazol  4-
yloxy)  3-[2-(2 –methoxyphenoxy) ethylamino] propan-2-ol
for CAR, 2 – hydroxyl  5-[1-hydroxyl  2-(4-phenylbutan
 2-ylamino) ethyl] benzamide. HCl for LAB, 1-(6-fluoro-3,
4-dihydro-2H-chromen-2-yl) 2-[[2-(6-fluoro-3, 4-dihydro-
2H-chromen-2-yl) 2-hydroxyethyl] amino] ethanol. HCl for
NEB, 1-naphthalen-1-yloxy-3-(propan-2-ylamino) propan-2-
ol for PRO, N-[4-[1-hydroxy  2-(propan  2-ylamino)
ethyl] phenyl] methanesulfonamide. HCl for SOT and (2S)-1-
(tert-butylamino)-3-[(4-morpholin-4-yl-1, 2, 5-thiadiazol-3-yl)
oxy] propan  2 – ol .maleate for TIM (Figure 1).
14 A. F. ABDEL HAKIEM ET AL.
Figure 1. Structures of the studied Beta-blockers.
Authentic samples of the studied drugs; ATE, BIS, BET,
CAR, LAB, NEB, PRO, SOT and TIM were kindly intro-
duced freely by a number of local and international pharma-
ceutical companies working in Egypt, AstraZeneca (6th
October city, Giza, Egypt), Global Napi Pharmaceuticals
(6th October city, Giza), Borg Pharmaceutical Industries
(Mokatam, Cairo, Egypt), Multi-Apex Pharma (Badr city,
Cairo, Egypt), Aldbeky Pharmaceutical Industries (El-Obour
city, Cairo, Egypt), Marcyrl Pharmaceutical Industries (El-
Obour city, Cairo, Egypt), AstraZeneca (6th October city,
Giza, Egypt), Amoun Pharmaceutical Company (El-Obour
city, Cairo, Egypt) and Egyptian Pharmaceutical Company
(EIPICO) (10th of Ramadan, Elsharqiya, Egypt), respectively.
Methanol and chloroform were purchased from Sigma-
Aldrich (St. Luis, USA). Ammonia solution, glacial acetic
acid, liquid paraffin oil, n-hexane and methylene chloride
were provided of El Nasr company for medicinal chemicals
(Abou Zaabal-El Qalyubia – Egypt). All solvents, chemicals
and reagents were of analytical grade.
Experimental
Sample solutions
Accurately weighed 100.0 mg amounts of each drug into
100-mL volumetric flasks and made to the mark with
methanol to get working solutions of 1.0 mg/mL
concentrations.
Chromatographic system preparation
Pre-coated TLC silica gel aluminum plates 60 F254
(20  20 cm, 200 mm thickness, Merck KGaA, 64271
Darmstadt, Germany) were used. They were activated firstly
by immersing in methanol solution for few seconds followed
by air drying. Imparted lipophilic characters were applied by
impregnation method for 30 seconds into 5% liquid paraffin
oil in methylene chloride[22] and n-hexane[23] followed by air
drying. This allows getting two kinds of reversed phases of dif-
ferent lipophilicities in addition to the untreated normal phase
ones. Chromatographic behaviors of analytes on each station-
ary phase were developed by four mobile phase systems of
varying polarities and pH values, CH3OH (3.5)–CHCl3
(7.5)–HOAC 0.2% (v/v), CH3OH (3.5)–CHCl3 (7.5)–NH4OH
0.2% (v/v), CH3OH (7.5)–CHCl3 (3.5)–HOAC 0.2% (v/v),
CH3OH (7.5)–CHCl3 (3.5)–NH4OH 0.2% (v/v) are considered
as systems A, B, C and D, respectively.
The prepared eluent systems were then poured into TLC
glass tanks (Sigma-AldrichV Co., USA) lined in their edges
R
with filter paper to enhance chambers saturation and cov-
ered with tight lids for thirty minutes to allow saturation of
solvent mixtures before every run.
Chromatography
Samples were applied as bands (4.0 mm width) by spotting
of 5.0 lL of each working solution by means of CAMAG
Linomat V automatic sample applicator (CAMAG, Muttenz,

Switzerland) fitted with a 100.0 lL syringe (Linomat syringe
659.004, Hamilton-Bonaduz Schweiz, CAMAG). Plates are
then dipped into the pre-saturated mobile phase chambers.
Scanning was carried out by UV lamp of short wavelength
254.0 nm (VL-6.LC, MARNE LA VALLEE Cedex 1, France)
at room temperature.
Computational modeling
Statistical methodologies are exploited to detect if created
models are sound and unbiased. The machine learning (ML)
computer programming (MOE 2019.01 software)[24] was
used in statistical and mathematical calculation of data using
partial least square (PLS) and multiple linear regression
(MLR) techniques. It utilizes multivariate methods to model
relevant properties as a function of chromatographic behav-
ior of nine structurally related beta-blockers. The investiga-
tion included 315 molecular descriptors calculated by the
program for each drug. Using the contingency analysis[25]
approaches embedded in the program, a set of the most
important molecular descriptors has been chosen for each
TLC system used. Validation of the established models has
been developed utilizing data created by the models in what
is called internal method. The applied software is exploiting
many approaches of internal validation.
The least squares fitting is the most common internal valid-
ation model. It resembles linear regression and utilized for
comparison between the experimental and predicted activities.
It is considered as an improved method of determining R2,
Equation (3), in order to give a robust straight line fit, where
data points are far from the central data points (essentially
data points a specified standard deviation away from the
model) are given less weight when calculating the R2.
2 X X X 
N XY  X Y
6 ffi7
R2 ¼ 6rffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 7
4 h X 2 X 2 ih X 2 X 2 i5
N X  X N
where N represents consecutive blocks of samples. Y and X
are the predicted and experimental RM values, respectively.
Cross validation (CV) which measures how large the applic-
ability of model for a given data set, has been performed by
repeated regressions on data subsets by excluding each mol-
ecule once (leave one out) and sometimes by more than one
molecule (leave many out, LMO), in turn, the correlation is
measured using the predicted values of the missing molecule/s.
The prediction statistics of the final model are deter-
mined by the correlation coefficient of multiple determin-
ation (R2) Equation (4) (known also as multiple correlation
coefficient and explained variance in fitting), calculated for
the training set.
X moieties, two chroman moieties and morpholine and thia-
R2 ¼ 1  ðyei  yci Þ2
X i (4)
ðyei  hye iÞ2
i
JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 15
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 from calibration.
The root-mean squared error (RMSE) Equation (5),
which exhibits error between the mean of the experimental
values and predicted ones was applied for fitting of the
developed QSRR models, to explore the reliability of models
if they possess predictive quality reflected on numerical val-
ues of correlation coefficients.
!
Xn
ð^y i  ym Þ2
RMSE ¼ Sqrt (5)
i¼1
n1
where y and ^y are the experimental and predicted RM values
for an individual compound in the training set, ym is the
mean of the experimental RM values, and n is the number of
molecules in the set of data being examined.[26,27]
Results and discussion
Retention behavior of analytes
Regarding normal phase, the investigated drugs showed
varying retardation behaviors in accordance to their molecu-
lar structures as well as natures of used mobile phase sys-
tems. In system A, Atenolol, CAR and PR showed tendency
to the moderately nonpolar system giving retardation factor
(RF) of 0.30, 0.75 and 0.67, respectively. This could be attrib-
uted to retarded ionization of silanol groups as well as
protonation provided by glacial acetic acid to both the side
chain secondary amino group and the primary one of aceta-
mide moiety of ATE that imparted sufficient polarity to be
eluted with the slightly polar mobile phase system. Similarly,
the secondary amino and heterocyclic nitrogen of carbazole
32 moiety in CAR and the secondary amino group of PRO
were subjected to protonation but the imparted polarity was
unable to overcome the high lipophilicity of either carbazole
Y  Y and methoxyphenoxy moieties or naphthalene moiety of
CAR and PRO, respectively, hence they remain un-eluted.
(3)
Bisoprolol fumerate, BET, LAB, NEB and TIM show large
RF values because of their decreased or diminished ioniza-
tion in this acidic medium since they are represented as
fumerate, hydrochloride and maleate salts, respectively. In
system B, BIS and BEX salts were ionized, ATE was also
subjected to ionization of its side chain hydroxyl moiety.
Their strong attraction to the ionized stationary phase (i.e.,
ionization of silanol moieties) since the imparted polarities
made them unable to be carried by the mostly nonpolar elu-
ent system. Labetalol HCl, NEB and TIM salts were ionized
in addition to ionization of their side chain hydroxyl moi-
eties hence to some sort repulsion with the ionized station-
ary phase as well as their lipophilic criteria of their large
hydrocarbon structures (i.e., phenylbutane and benzamide
diazole moieties of LAB, NEB and TIM, respectively)
improved their affinities to the mostly nonpolar mobile
phase system, giving large RF values. In different manner,
the polar character of SOT by its sulfacetamide moiety
16 A. F. ABDEL HAKIEM ET AL.

Table 1. RF and RM values of analytes on studied stationary phases using different eluent systems.
Eluent systems ATE BIS BET CAR LAB NEB PRO SOT TIM
Normal Phase
CH3OH (3.5)–CHCl3 (7.5)–HOAC 0.2 % (v/v) 0.3100 0.7100 0.7800 0.7500 0.6600 0.7800 0.6700 – 0.73
0.3475 0.3888 0.5497 0.4771 0.2881 0.5497 0.3076 – 0.4319
CH3OH (3.5)–CHCl3 (7.5)–NH4OH 0.2 % (v/v) – – – 0.9000 0.6800 0.8200 0.6900 0.4500 0.7800
– – – 0.9542 0.3274 0.6585 0.3475 0.0871 0.5497
CH3OH (7.5)–CHCl3 (3.5)–HOAC 0.2 % (v/v) – – 0.7200 0.8300 0.8500 – 0.7700 – 0.7900
– – 0.4102 0.6886 0.7533 – 0.5248 – 0.5754
CH3OH (7.5)–CHCl3 (3.5)–NH4OH 0.2 % (v/v) 0.5200 0.7200 0.6200 0.8900 0.7800 0.8500 0.6800 0.6200 0.7500
0.0348 0.4102 0.2126 0.9080 0.5497 0.7533 0.3274 0.2126 0.4771
5 % Paraffin Oil in CH2Cl2 Phase
CH3OH (3.5)–CHCl3 (7.5)–HOAC 0.2 % (v/v) 0.2600 – – – 0.5500 – 0.5600 0.4200 0.5900
0.4543 – – – 0.0871 – 0.1047 0.1402 0.1581
CH3OH(3.5)–CHCl3 (7.5)–NH4OH 0.2 % (v/v) 0.3300 – – – 0.5400 – 0.5800 0.4200 0.6500
0.3076 – – – 0.0696 – 0.1402 0.1402 0.2688
CH3OH (7.5)–CHCl3 (3.5)–HOAC 0.2 % (v/v) – – – – – – – – –
– – – – – – – – –
CH3OH (7.5)–CHCl3 (3.5)–NH4OH 0.2 % (v/v) 0.4600 0.6100 – 0.8500 0.6700 0.7800 0.5600 0.5100 0.6700
0.0696 0.1943 – 0.7533 0.3076 0.5497 0.1047 0.0174 0.3076
5 % Paraffin Oil in n-Hexane Phase
CH3OH (3.5)–CHCl3 (7.5)–HOAC 0.2 % (v/v) 0.2700 0.6000 – 0.6500 0.5700 – 0.5500 0.4200 0.6500
0.4320 0.1761 – 0.2688 0.1224 – 0.0871 0.1402 0.2688
CH3OH(3.5)–CHCl3 (7.5)–NH4OH 0.2 % (v/v) 0.3100 0.7200 0.7400 0.7700 0.6000 – 0.6600 0.5000 –
0.3474 0.4102 0.4543 0.5247 0.1761 – 0.2881 – –
CH3OH (7.5)–CHCl3 (3.5)–HOAC 0.2 % (v/v) – – – 0.7500 0.7300 – 0.6600 0.7300 –
– – – 0.4771 0.4312 – 0.2881 0.4320 –
CH3OH (7.5)–CHCl3 (3.5)–NH4OH 0.2 % (v/v) 0.4200 0.6100 0.6100 0.8700 0.7100 – 0.6100 0.5900 0.7100
0.1402 0.1943 0.1943 0.8256 0.3889 – 0.1943 0.1581 0.3889
Values at blank cells represent RF, while values at blue highlighted cells represent RM.

retarded its elution to certain extent. The Imparted polarity
of both ionized BIS and BEX salts inhibited their chroma-
tographic elution. The highly lipophilic structure of CAR
and PRO as well as the ionized stationary phase provoked
their affinity to the non-polar eluent hence showed much
higher RF values. Concerning system C, the polar station-
ary phase remains unionized. The imparted protonation of
ATE, BIS, NEB and SOT was unable to overcome the lipo-
philic characters of analytes in addition the decreased
probability for hydrogen bonding with the mostly polar
mobile phase system, hence they showed great attraction
to stationary phase and remain un-eluted. At the same
time, protonation enhances polarity of BET, CAR, LAB,
PRO and TIM showing attraction to the polar eluent.
Alkalinity of system D caused variable ionization of differ-
ent investigated salts as well as side chain hydroxyl moi-
eties improving their polar criteria and hence, they
exhibited different mobilities in the polar mobile phase.
The imparted lipophilic character to stationary phase by
5% liquid paraffin in methylene chloride solution was
more that obtained by its solution in normal hexane. This
is evidenced by elution inhibition of many analytes in
acidic media of system A and especially retardation of all
drugs in system C as they almost unionized and imparted
protonation didn’t provide sufficient polarity to be
attracted to the fairly or mostly polar eluent systems A
and C, respectively. Greater lipophilicity imparted by paraf-
fin oil in methylene chloride also obviously explored in
basic media of system B and D, and presented as more
strong retardations of analytes (i.e., smaller or no RF val-
ues), Table 1.
Chemometric data analysis
Partial least square regression analysis in MOE software was
used to correlate and establish relationship between, X as
latent variables (molecular descriptors) and Y which repre-
sents RM as dependent variable, by a linear multivariate
model. Additionally, simple linear regression analysis has
been established between Y (predicted RM) and X (experi-
mental RM). Among advantages of PLS, it can analyze data
involving many, noisy, collinear, and even incomplete varia-
bles in both X and Y matrices, and hence, it is more con-
venient than MLR. Additionally, other than the number of
investigated analytes and molecular descriptors, it possess
great predictive ability enabling establishing reliable QSRR
models as well as easy increasing number of relevant varia-
bles to improve precisions of models.[28]
Before the analysis, the data should be standardized in
order to eliminate the impact of different scales in the devel-
oped methods for lipophilicity determination, the analyzed
data were mean-centered and scaled to unit variance.[29,30]
The variables (descriptors) were normalized or recalculated
by using Z-score scaling algorithm (V ¼ mean of V/d, where
V is the value of variables and d is the standard deviation)
then scaled. For feature descriptors, 0.0 values indicate the
feature is absent.[31,32] In the present study, all calculated val-
ues are above zero and ranged from 0.0328 to 2.3091, and
not exceeding 2.5 indicating that there are no outliers [33],
furthermore minimal residuals (RES) obtained (difference
between predicted value and experimental ones), Table 2
indicating that all investigated descriptors are featured.
Models have been developed for four mobile phase sys-
tems for each studied stationary phase. It is worth noting
 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 17

Table 2. Input normalization and scaling.

Stationary phase Eluent system Analyte RM(PRED)a RESb Z-SCOREc
Normal phase CH3OH (3.5)–CHCl3 (7.5)–HOAC 0.2 % (v/v) ATE 0.2060 0.1417 0.9757
BIS 0.3841 0.0047 0.0328
BET 0.6366 0.0869 0.5986
CAR 0.4596 0.0174 0.1202
LAB 0.3445 0.0564 0.3882
NEB 0.4019 0.1478 1.0174
PRO 0.4103 0.1028 0.7076
SOT 0.1176 0.1176 0.8094
TIM 0.0965 0.3354 2.3091
CH3OH (7.5)–CHCl3 (3.5)–HOAC 0.2 % (v/v) ATE 0.2068 0.2068 1.4572
BIS 0.0440 0.04377 0.3084
BET 0.2216 0.2216 1.5616
CAR 0.8132 0.1410 0.9937
LAB 0.4896 0.1623 1.1435
NEB 0.6406 0.0178 0.1261
PRO 0.2008 0.1466 1.0332
SOT 0.0441 0.1313 0.9253
TIM 0.5030 0.0466 0.3286
CH3OH (7.5)–CHCl3 (3.5)–NH4OH 0.2 % (v/v) ATE 0.0135 0.0212 0.2422
BIS 0.2538 0.1563 1.7840
BET 0.3945 0.1818 2.0757
CAR 0.8545 0.0534 0.6103
LAB 0.5702 0.0205 0.2346
NEB 0.7025 0.0507 0.5794
PRO 0.3906 0.0632 0.7219
SOT 0.1967 0.0158 0.1814
TIM 0.5091 0.0319 0.3651
5 % Paraffin oil in CH2Cl2 CH3OH (3.5)–CHCl3 (7.5)–HOAC 0.2 % (v/v) ATE 0.7320 2.7487 0.4368
BIS 2.2640 0.0060 0.0721
BET 2.6980 0.0060 0.0911
CAR 3.9750 1.0451 0.0205
LAB 2.5640 0.1661 0.0594
NEB 3.2150 1.0631 0.0566
PRO 2.9210 0.0061 0.1010
SOT 0.7320 2.7487 0.4368
TIM 2.2640 0.0060 0.0721
CH3OH (7.5)–CHCl3 (3.5)–HOAC 0.2 % (v/v) ATE 0.3268 0.0192 0.4275
BIS 0.0351 0.0351 0.7810
BET 0.0810 0.0810 1.7897
CAR 0.0125 0.0125 0.2785
LAB 0.0665 0.0031 0.0678
NEB 0.0670 0.0670 1.4868
PRO 0.1040 0.0362 0.8045
SOT 0.0960 0.0443 0.9843
TIM 0.2220 0.0471 1.0464
CH3OH (7.5)–CHCl3 (3.5)–NH4OH 0.2 % (v/v) ATE 0.1580 0.0883 0.8745
BIS 0.0585 0.1360 1.3440
BET 0.1941 0.1941 1.9212
CAR 0.6405 0.1130 1.1171
LAB 0.3766 0.0691 0.6837
NEB 0.4995 0.0501 0.4961
PRO 0.1858 0.0811 0.8025
SOT 0.0242 0.0068 0.0673
TIM 0.3436 0.0361 0.3570
5 % Paraffin oil in n-Hexane CH3OH (3.5)–CHCl3 (7.5)–HOAC 0.2 % (v/v) ATE 0.3951 0.0370 0.3901
BIS 0.1850 0.0090 0.0940
BET 0.1144 0.1144 1.2110
CAR 0.1615 0.1074 1.1360
LAB 0.2637 0.1413 1.4950
NEB 0.0240 0.0240 0.2544
PRO 0.0115 0.0756 0.7995
SOT 0.1170 0.0233 0.2470
TIM 0.1030 0.1660 1.7560
CH3OH (7.5)–CHCl3 (3.5)–HOAC 0.2 % (v/v) ATE 0.3250 0.0225 0.1795
BIS 0.2540 0.1564 1.2460
BET 0.4998 0.0456 0.3632
CAR 0.3403 0.1844 1.4701
LAB 0.2182 0.0421 0.3360
NEB 0.2790 0.2790 2.2210
PRO 0.2844 0.0040 0.0292
SOT 0.0123 0.0122 0.0974
TIM 0.0323 0.0323 0.2572
CH3OH (7.5)–CHCl3 (3.5)–NH4OH 0.2 % (v/v) ATE 0.1167 0.2570 1.8292
BIS 0.1791 0.0151 0.1080
(continued)
18 A. F. ABDEL HAKIEM ET AL.

Table 2. Continued.
Stationary phase Eluent system Analyte RM(PRED)a RESb Z-SCOREc
BET 0.1800 0.0142 0.1012
CAR 0.8512 0.0260 0.1826
LAB 0.1561 0.2330 1.6572
NEB 0.1553 0.1553 1.1062
PRO 0.2265 0.0323 0.2295
SOT 0.1230 0.0354 0.2520
TIM 0.2160 0.1727 1.2295
a
Predicted value calculated by MOE software.
b
Difference between the value of the model (predicted value) and the activity field (experimental value, RM).
c
Absolute difference between the value of the model calculated by MOE software and the activity field (RM), divided by the square root of the mean square error
of the dataset.

Table 3. Values of the best correlated molecular descriptors and their values with respect to the studied analytes.
Molecular Descriptors
Analytes logP(o/w) Log S E_str h_pstrain PEOE_VSA þ 6 PEOE_PCþ vsurf_DD23
ATE 0.7320 1.9677 0.7313 2.7490 0.0000 1.9966 0.7071
BIS 2.2640 2.5430 0.8099 0.0060 17.2380 3.1133 1.1180
BET 2.6980 2.4361 1.0405 0.0060 17.2380 2.8120 1.0000
CAR 3.9750 5.0313 1.8524 1.0451 0.0000 2.5942 9.9750
LAB 2.5640 3.1470 1.5991 0.1661 17.2380 3.2402 0.7071
NEB 3.2150 4.0031 1.7192 1.0631 0.0000 2.4360 0.5000
PRO 2.9210 3.5076 0.9402 0.0061 17.2380 2.4815 1.5811
SOT 1.0120 1.3910 1.1460 0.5624 17.2380 2.6540 0.7071
TIM 1.0340 1.4926 2.0350 0.0056 44.1843 3.2260 2.0000
logP(o/w) Log Octanol/water partition coefficient
Log S Log Solubility in water
E_str Bond stretch energy
h_pstrain Strain energy to recover input protonation state
PEOE_VSA þ 6 Total positive 6 vdw surface area
PEOE_PCþ Total positive partial charge
vsurf_DD23 vsurf _EDmin2. vsurf _EDmin3 distance

Table 4. Two molecular descriptors validated linear regression models.

Stationary phases Eluent systems Estimated linear models R2 Root mean square error (RMSE)
Normal phase CH3OH (3.5)–CHCl3 (7.5) RM ¼  0.04731  0.50377 logP (o/w)  0.31602 logS (6) 0.72494 0.14526
–HOAC 0.2 % (v/v)
CH3OH (3.5)–CHCl3 (7.5) RM ¼ 0.68230  0.13729 logP (o/w)  0.51277 E_str (7) 0.82595 0.14192
–NH4OH 0.2 % (v/v)
CH3OH (7.5)–CHCl3 (3.5) RM ¼ 0.34273  0.13905 logP (o/w)  0.34798 E_str (8) 0.88695 0.08762
–NH4OH 0.2 % (v/v)
5 % Paraffin oil in CH3OH (3.5)–CHCl3 (7.5) RM ¼ 0.02598  0.04375 logP (o/w) þ 0.16112 h_pstrain (9) 0.83898 0.06858
CH2Cl2 –HOAC 0.2 % (v/v)
CH3OH (3.5)–CHCl3 (7.5) RM ¼ 0.40342  0.10465 logP(o/w)  0.01170 PEOE_VSA þ 6 (10) 0.91345 0.04504
–NH4OH 0.2 % (v/v)
CH3OH (7.5)–CHCl3 (3.5) RM ¼ 0.50863  0.12305 logP (o/w)  0.35631 E_str (11) 0.84484 0.10102
–NH4OH 0.2 % (v/v)
5 % Paraffin oil in CH3OH (3.5)–CHCl3 (7.5) RM ¼ 1.22837  0.10157 logP (o/w)  0.38012 PEOE_PCþ (12) 0.79419 0.09452
n-Hexane –HOAC 0.2 % (v/v)
CH3OH (3.5)–CHCl3 (7.5) RM ¼ 0.08794  0.49181 logP (o/w)  0.30340 logS (13) 0.77610 0.12547
–NH4OH 0.2 % (v/v)
CH3OH (7.5)–CHCl3 (3.5) RM ¼  0.05018  0.02155 logP (o/w)  0.07172 vsurf_DD23 (14) 0.70525 0.14042
–NH4OH 0.2 % (v/v)
The standard deviation of residuals of regression line.

that, since the worse correlation models obtained with elu-
ent system 3, all corresponding results were excluded. The
developed models described relations of molecular structures
of studied drugs and the best correlated descriptors (these
descriptors are identified in Table 4) in terms of their chro-
matographic behaviors represented as RM values. These
descriptors are; logP(o/w), log S, E_str, h_pstrain,
PEOE_VSA þ 6, PEOE_PC þ and vsurf_DD23. The logP(o/
w) parameter as a measure of lipophilicity is kept as a
potential and basic descriptor in all developed multivariate
correlations because of its importance in drug pharmacokin-
etics and discovery as well as it is one of the main determi-
nants for drugs retentions on different stationary phases.[34]
The obtained numerical values of the utilized molecular
descriptors by different models are illustrated in Table 3.
Good correlations were obtained by two descriptors CV
models with R2 values above the minimum acceptable value
of 0.60.[35,36] The R2 values are varying in range of
0.70525  0.91345, Table 1, indicating reliable regression sta-
tistics. The best R2 values (average of 0.86575) emerge with
 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 19

Figure 2. 3 D plots for the two parameters correlation between RM and the investigated molecular descriptors. Numbers from 1 to 9 represent ATE, BIS, BET, CAR,
LAB, NEB, PRO, SOT and TIM, respectively. (A), (B) and (C) represent investigated stationary phases; normal phase, reversed phase imparted by impregnation of nor-
mal phase into 5% paraffin oil solution in methylene chloride and n-hexane, respectively. I, II, and III represent elution systems 1, 2 and 4, respectively.

reversed phase of 5% paraffin oil in methylene chloride indi-
cating the high dependency of RM on E_str, h_pstrain and
PEOE_VSA þ 6 representing bond stretch energy, strain
energy to recover input protonation state and the total vdw
surface area. A much lesser correlation (average of 0.75851)
is exhibited between chromatographic retardation of studied
analytes and their RM values with reversed phase imparted
by paraffin oil solution in n-hexane. Another set of descrip-
tors affected chromatographic behavior of analytes, namely
PEOE_PCþ, logS and vsurf_DD23 representing total posi-
tive partial charge, log solubility in water and vsurf
_EDmin2. vsurf _EDmin3 distance, respectively. The so far
difference in R2 values of the formulated reversed phases
reveals the great effect of dissolving solvent in manipulating
20 A. F. ABDEL HAKIEM ET AL.
Figure 3. Correlation between experimentally obtained RM values and calculated ones in different studied chromatographic systems. (A), (B) and (C) represent the
obtained linear regressions on investigated stationary phases; normal phase, reversed phase imparted by impregnation of normal phase into 5% paraffin oil solution
in methylene chloride and n-hexane, respectively.
the lipophilic character. The obtained multiple correlation
coefficient (average of 0.81261) of normal phase lie in mid-
dle range between the two investigated reversed phases
depending on logS and E_str descriptors. To the best of our
knowledge, that relatively excellent correlation of 0.91345 is
obtained with eluent system 2 on imparted reversed phase
by paraffin oil solution in methylene chloride. Acceptable
low values of root mean square error (RMSE) as one of
applied internal validation approaches by software were
obtained. It ranges from 0.04504 to 0.14526 confirming
method reliability (Table 4).
The three dimensional plot of normal phase
(Figure 2A(I)) indicates clustering of ATE and LAB in elu-
ent system 1 owing to relative resemblance in their polarities
because of quiet structural features similarity represented in
phenoxypropyl and acetamide moieties in ATE correspond-
ing to hydroxyl phenyl and benzamide moieties in LAB as
well as ionization retardation of LAB HCl in acidic medium
of glacial acetic acid. This behavior is repeated on reversed
phase imparted by paraffin oil solution in methylene chlor-
ide and n-hexane by eluent system 1 and eluent system 2
although encouraged ionization of LAB salt in basic medium
of ammonia, this could be much less effective in comparison
with structural similarities (Figure 2B(I and II), 2C(II)).
Clustering of both PRO and BIS is attributed to that polarity
imparted by ethoxymethyl and phenoxy moieties in BIS are
quite equivalent to that of naphthalene moiety of PRO in
addition to structural similarities of propan-2-ylamino and
propan 2-ol moieties as well as diminished ionization of
BIS fumarate salt in glacial acetic acid medium. Benzamide,
flourochromen, methanesulfonamide, morpholin and thia-
diazole moieties impart relatively similar polarities to LAB,
NEB, SOT and TIM, respectively resulting in gathering of
their 3 D representations on normal phase upon develop-
ment by eluent system 4 (Figure 2A(III)) and in addition to
the ionization effect in basic medium of eluent system 2
 JOURNAL OF LIQUID CHROMATOGRAPHY & RELATED TECHNOLOGIES 21

Table 5. Linear regression analysis between predicted retentions and experimental ones in different investigated chromatographic systems.
Correlation
Stationary phases Eluent systems Estimated linear models coefficient (R2)
Normal phase CH3OH (3.5)–CHCl3 (7.5) RM (Pred.) ¼ 0.74542 RM (Exp.)  0.06948 (15) 0.7081
–HOAC 0.2 % (v/v)
CH3OH (3.5)–CHCl3 (7.5) RM (Pred.) ¼ 0.751472 RM (Exp.)  0.10415 (16) 0.7098
–NH4OH 0.2 % (v/v)
CH3OH (7.5)–CHCl3 (3.5) RM (Pred.) ¼ 0.88695 RM (Exp.)  0.048808 (17) 0.8869
–NH4OH 0.2 % (v/v)
5 % Paraffin oil in CH3OH (3.5)–CHCl3 (7.5) RM (Pred.) ¼ 0.819312 RM (Exp.) þ 0.0260547 (18) 0.9186
CH2Cl2 –HOAC 0.2 % (v/v)
CH3OH (3.5)–CHCl3 (7.5) RM (Pred.) ¼ 0.915215 RM (Exp.) þ 0.0117452 (19) 0.9800
–NH4OH 0.2 % (v/v)
CH3OH (7.5)–CHCl3 (3.5) RM (Pred.) ¼ 0.925354 RM (Exp.) þ 0.00406141 (20) 0.9065
–NH4OH 0.2 % (v/v)
5 % Paraffin oil in CH3OH (3.5)–CHCl3 (7.5) RM (Pred.) ¼ 0.810075 RM (Exp.) þ 0.0102567 (21) 0.8353
n-Hexane –HOAC 0.2 % (v/v)
CH3OH (3.5)–CHCl3 (7.5) RM (Pred.) ¼ 0.84187 RM (Exp.) þ 0.0011815 (22) 0.9080
–NH4OH 0.2 % (v/v)
CH3OH (7.5)–CHCl3 (3.5) RM (Pred.) ¼ 0.74810 RM (Exp.)  0.049973 (23) 0.7199
–NH4OH 0.2 % (v/v)

Figure 4. Principle component analysis scores of experimental RM values.

(Figure 2A(II)). For the same reasons, LAB, NEB and SOT,
TIM are exhibited in nearby positions, Figure 2B(II), and
similarly, LAB, NEB and SOT, Figure 2B(III). The Nearby
positioning of ATE and TIM on the formulated reversed
phases in the mostly polar and mostly non-polar basic elu-
ent systems 2 and 4 is attributed to ionization of hydroxyl
moieties and maleate salt of TIM as well as quiet similar
polarities by propoxy and phenyl] acetamide moieties and
morpholin and thiadiazol moieties of ATE and TIM,
respectively (Figure 2B(II),2C(II)). The obvious deviation of
BET, CAR and PRO, since have the most hydrophobic char-
acters among studied compounds with log p values of 4.23,
3.10 and 2.66, respectively. Additionally, both BET and CAR
has shown far deviation. Concerning BET, this could be
attributed to the steric arrangement of the cyclopropyl
methyl group and the possible interactions of the ether oxy-
gen with utilized solvents. interacted strongly with the non-
polar stationary phase, but in case of CAR, its lipophilicity
was strongly affected its high pKa of 7.9. Logically, other
analytes have exhibited some sort of gathering since they
possess close log P as well as pKa values.[37]
The prediction capabilities of the developed models in
different investigated chromatographic conditions specified
by Equations (6–14), Table 4 are represented by plotting the
respective experimental RM values against the calculated
ones (Figure 3). Reliable linear regressions were obtained of
correlation coefficient (R2) range 0.70810.9800, Table 5.
This indicates satisfactory fitting to experimental data.
Closeness of slopes to unity, reveals that the method is free
from proportional error. Additionally, intercepts values are
almost zero proving the absence of systematic error and
method bias.
This reflects the efficient prediction power of the devel-
oped models and hence the proposed models can correctly
represent the relationship between the retention parameters
of the investigated structurally related analytes on normal
22 A. F. ABDEL HAKIEM ET AL.
Figure 5. Principle component analysis scores of predicted RM values.
Figure 6. Principle component analysis scores of selected molecular descriptors values.
phase and the two formulated reversed phases developed by
different eluent systems and the molecular descriptors calcu-
lated solely from their molecular structures. In accordance,
these models are proven to predict retention of the studied
structurally similar beta blockers under the same chromato-
graphic conditions.
An accurate estimation of resemblance in experimental
and predicted chromatographic behavior of studied analytes
as well as dependency on best correlated descriptors was
assessed by principle component analysis (PCA) using
MATLAB software.[38] Matrices of the available RM values
with respect to analytes for establishing the PCA models.
The obtained scoring (Figures 4 and 5) evidenced variation
between experimental chromatographic behavior and pre-
dicted one of almost all analytes, and this is attributed to
that they show variable strengths of correlations between
experimental and predicted RM values in investigated chro-
matographic systems. Regarding experimental data, close
inspection of Figure 4, reveals a proper closeness in chroma-
tographic behavior of LAB and TIM. Five analytes; LAB,
TIM, PRO, BIS and BET lie in a confined area representing
relatively moderate similarities in their retardations.
Atenolol, NEB and CAR exhibit so far and distinct chroma-
tographic behaviors among all analytes. Investigation of pre-
diction scoring, Figure 5, shows deviation of CAR, PRO and
SOT at the same time, aggregation of the rest of the analytes
in an area of mild similarity with relatively close behaviors
of BET, NEB and BIS, TIM. The values concerning each
analyte of the best correlated descriptors obtained after care-
ful contingency analysis of 315 descriptors were devoted for
establishing matrix for PCA. The obtained scoring, Figure 6,
is useful and good indication for structural dependency on
the selected descriptors. Sotalol HCl, PRO, LAB, BIS and
BET exhibit relatively equitable dependency with very high
matching between BIS, BET and LAB represented by over-
lapping corresponding dots. On the other hand, ATE and

NEB show very close dependency but far from the previ-
ously mentioned set of analytes. Furthermore, CAR and
TIM presented totally different dependency than
other analytes.
Conclusion
The lipophilicity and retention behavior of nine structurally
related beta-blockers were investigated on three stationary
phases; normal phase and two formulated reversed phases
by impregnation technique of normal phase plates into 5%
solutions of paraffin oil in methylene chloride and n-hexane.
It was found that solvent variation resulted in reversed
phases of different lipophilicities. The effect of four develop-
ment systems varying in polarity and pH was studied. The
QSRR modeling was performed using partial least square
regression, in order to select the most important variables
that describe the behavior of the investigated compounds.
The established two-parameter models show satisfactory pre-
dictive power and correlation. This is clearly explored by the
good correlation between the experimental and predicted
retentive behaviors of analytes upon utilizing logP(o/w), log
S, E_str, h_pstrain, PEOE_VSA þ 6, PEOE_PC þ and
vsurf_DD23 as molecular descriptors. The developed
internal validation study evidenced the high significant cor-
relation between retention and these descriptors. Hence
might be exploited to expect retentions of the investigated
beta blockers with a considerable degree of confidence in
rather difficult experimental determinations or those sub-
jected to large uncertainties. This study gives a quantitative
assessment of the relative importance of specific chromato-
graphic molecular interactions.
ORCID
Ahmed Faried Abdel Hakiem http://orcid.org/0000-0002-1551-1124
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