(2024) 18:160
Mohamed et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01260-w

BMC Chemistry

RESEARCH

Open Access

Independent concentration extraction
as a novel green approach resolving overlapped
UV–Vis binary spectra and HPTLC‑densitometricmethods for concurrent determinationof levocloperastine and chlorpheniramine
Ekram H. Mohamed1, Hany A. Batakoushy2*, Adel Ehab Ibrahim3,4, Zeinab Adel Nasr5*, Marwa M. Soliman5,
Sona S. Barghash5, Tahany F. Mohamed5 and Fatma A. Fouad5

Abstract
Background The proposed research study introduces independent concentration extraction (ICE) as a novel
UV–Vis spectrophotometric approach. The approach can be used for extracting the concentration of two analytes
with severely overlapped spectra from their binary mixtures. ICE is based on spectral extraction platform involving
simple smart successive methods that can directly extract the original zero order spectra of the analytes at theircharacteristic (λmax). Chlorpheniramine maleate (CPM) and Levocloperastine fendizoate (LCF) are two commonly coformulated drugs in cough preparations. The combined mixture was used to confirm the validity of the developed ICE
tool. Another less green HPTLC was developed for the first time to separate both drugs and help also in confirming
the proposed tool.
Methods For the simultaneous determination of CPM and LCF, two ecologically friendly techniques were employed.
The first approach encompasses the use of the ICE spectrophotometric method that could be successively applied
for extracting the concentration of two analytes with severely overlapped unresolved spectra in their binary mixtures.
Other complementary methods aiming at original spectral extraction; including spectrum subtraction (SS) and unity
subtraction (US) were also successfully employed to resolve the zero order spectra of the combined drugs with all
their characteristic features and peaks. The second technique used, a high-performance TLC-densitometric one,was performed on silica plates with silica plates F254 and a mobile phase with a ratio of 3:3:3:1 by volume of toluene,ethanol, acetone, and ammonia as a developing system at 230 nm.
Results The presented extraction approach was executed without any optimization steps or sample pretreatment for the simultaneous determination of CPM and LCF. The method was found to be valid for their determination within concentration range of 3.0–30.0 μg ­mL−1 for both drugs. For HPTLC method, the resulting ­Rfvalues of CPM and LCF were 0.37 and 0.78, within concentration ranges of 0.3–4.0 μg/spot and 0.8–10.0 μg/spot,*Correspondence:
Hany A. Batakoushy
hany.batakoushy@phrm.menofia.edu.eg
Zeinab Adel Nasr
zeinabadel@azhar.edu.eg
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Mohamed et al. BMC Chemistry(2024) 18:160

Page 2 of 16

respectively. Greenness assessment of both developed methodologies showed that the HPTLC method is less green
than the spectrophotometric method, yet with comparable sustainability when it comes to the used technique.
Conclusion The procedures were found to be selective, accurate, and precise for analysis of the studied binary mixture. Furthermore, the environmental impact of the introduced methods was assessed using novel greenness metrics,
namely AGREE and Green Analytical Procedure Index (GAPI) to prove their ecological safety. In addition, white analytical chemistry (WAC) evaluation metric was employed to ensure the synergy and coherence of analytical, practical,
and ecological attributes.
Keywords Green analytical chemistry, Spectral resolution, Independent concentration extraction, High performancethin layer chromatography, Chlorpheniramine, Levocloperastine

Background
The importance of pharmaceutical analysis (PA) is
increasing due to the globalization of pharmaceuticalmarket and enhanced concurrence between manufacturing companies where it is directly related to drug efficacy and safety. The main aim of PA is to develop, apply
and validate the good manufacturing practices (GMP)
to assure the production of safe pharmaceutical dosage
forms and products of standard high quality. This could
be achieved via tracking the concentration of active pharmaceutical ingredients throughout the whole manufacturing steps starting from purchasing the bulk powder or
its raw materials passing through the research and developments stages. Various instrumental methods of analysis could be used in PA. the selection of the optimum
method of analysis depend on many factors including thenature of compound under investigation, the complexityof the mixture, the underlying matrix, sample size, as wellas the analysis purpose [1].
Optical methods in general and UV–VIS spectroscopy in specific are the most convenient technique used
for routine analysis of active pharmaceutical ingredients
either due to their simplicity, cost effectiveness and theavailability of its appliances in nearly all quality control
laboratories. Moreover, UV spectroscopy methods arealso considered to be ecofriendly where safe solvents areusually used as water, ethanol or methanol beside low
wastes are generated. The specificity of the UV spectroscopic method was greatly improved by the introduction of smart resolution techniques [2–4], that enabled
the determination of multicomponent complex mixturesusing simple mathematical concepts without the need forany sophisticated or expensive instruments or programs.
Meanwhile, high performance thin layer chromatography (HPTLC) has gained a great momentum during thepast few decades especially with the advanced densitometric detection which is a more advanced and improved
version of the traditional TLC. HPTLC offers higher separation efficiency, improved resolution, and better reproducibility [5]. The application of HPTLC-densitometrictechnique was found to give fast and accurate results

while using very small sample amounts. In addition,
HPTLC-densitometric approach is cheap and doesn’t
need complicated procedures or instrumentation as
those required by HPLC [6].
Cough is a reflex action that helps to clear the throat
and airways of irritants, mucus, or foreign substances.
Cough could be brought on by acute and chronic respiratory disorders, as well as allergic conditions and asthma.
The body uses coughing, whether it is productive or dry,
to remove irritants from the airways and guard against
infection [7]. Several medication combinations are frequently used to treat coughs [8, 9]. Chlorpheniramine
maleate (CPM; chemical structure Fig. 1) is one of themost commonly used medications for the treatment ofallergic diseases [9]. Levocloperastine fendizoate (LCF;
chemical structure Fig. 1) is acting centrally as a coughsuppressant [10]. Combination of both medications thatis primarily used to treat dry cough [11, 12]. In such combination, CPM blocks the action of histamine, a chemicalthat triggers allergic reactions, and helps to relieve allergy
symptoms like itching, swelling, congestion, and stiffness.
Meanwhile, LCF blocks the transmission of nerve signals
from the cough center in the brain to the muscles thatcause coughing [7].
Reviewing literature showed various techniques that
had been developed to analyze CPM and LCF separately[13] or in their combination with other drugs [9, 14]. For

Fig. 1 Chemical structure of Levochlopersatine Fenodizoate (A)and Chlorpheniramine Maleate (B)Mohamed et al. BMC Chemistry(2024) 18:160

Page 3 of 16

the simultaneous analysis of both drugs, only few HPLC
methods have been reported [15, 16]. To our knowledge,
no UV–Vis spectrophotometry nor TLC-densitometry
methods have been yet reported for the simultaneous analysis of the drugs under study. The main objectives of this work are, firstly, to establish ICE approachand confirm its validation via simple eco-friendly steps
for the simultaneous determination of CPM and LCF in
bulk powder or in their combined dosage form. Anotheraim is to develop a novel green and sustainable HPTLCdensitometry method that could concurrently estimate
CPM and LCF. Moreover, the suggested methods wereassessed for their greenness using different tools including the green analytical procedure index (GAPI) [17],AGREE [18], and the white analytical chemistry [19] metrics which showed that the proposed approaches havehigh sustainability.

Theoretical concept for the novel
spectrophotometric approach

The amplitude difference (ΔPmix) is then computed where
the constant value resembling the concentration of N
would be cancelled and Δ ­Pmix is directly related to component M only as summarized in the following equation.
MPmix = P1 − P2 =1 + concentratin of N 1
nsN



M
−
2 + concentratin of N 2
nsN

(3)

Pmix =






M
M
1 −
2
nsN
nsN

The same amplitudes previously recorded at the selected
wavelengths(λ1 and λ2) are summed and the summation
value ( Pmix) is presented in the following equation.





M
1 + concentratin of N 1
Pmix = P1 + P2 =
nsN



M
+
2 + concentratin of N 2
nsN

(5)

Independent concentration extraction (ICE)

Independent Concentration extraction (ICE) is a new
simple method applied for the first time for the direct
estimation of analytes of interest with no need for any
other complementary method. It should be considered to
be an extension to the previously applied constant extraction (CE) method for determination of binary mixture
with overlapping [20]. The main difference between CE
and the proposed ICE is the use of the normalized spectra of drug of interest as a divisor. Thus, modulating theextracted constant directly to the concentration [21–23],
with no need to the constant multiplication step foundin CE method. The novel ICE method could be used toquantify two components (e.g., M and N) with complete
spectral overlap via only one main step depending on
complementary and successive mathematical equations
as summarized in the following scheme.
For determination of the first drug (N), as a divisor, thespectrum of the total binary mixture is divided by thenormalized spectra of M (nsN ). The obtained ratio spectrum could be expressed as following.

M + N ÷ nsN =

M
N
+
nsN
nsN

(1)

M + N ÷ nsN =M+ concentratin of N
nsN

(2)

The ratio spectrum’s amplitude is measured at two different wavelengths (λ1 and λ2), where the two drugs overlap.

(4)Pmix =







M
1 +
nsN



M
nsN




2 + 2 concentratin of N

(6)
Using the normalized spectrum of N ­(nsN) as a divisor
for different concentrations of M in its pure form, a direct
correlation is established between the amplitude difference(ΔPM) at the two preselected wavelengths and the summed
or total amplitude at the same wavelengths (­ PM Sum) and theregression equation is expressed as follows.
PM = Slope PM Sum + intercept


M
nsN







M
M
1 −
2 = Slope
1
nsN
nsN


M
+
2+ intercept
nsN

(7)

(8)

To calculate the concentration of component N, theamplitude values difference (ΔPmix) and sum (∑Pmix) at thetwo selected wavelengths on the ratio spectra, obtainedby dividing the total spectrum of the binary mixture by
ns N, were calculated as presented in Eqs. (4) and (6),respectively. The same calculated amplitude difference
was further manipulated to compute the ­(PM Sum) of purecomponent M from the corresponding regression Eq. (7).
The obtained ­(PM Sum) for pure M is then subtracted
from the (∑Pmix) and the concentration of N multiplied by
2 as summarized in the following equation.

Mohamed et al. BMC Chemistry(2024) 18:160

Page 4 of 16  
 

M
M
1 +
2
Pmix − PM Sum =nsN
nsN+ 2 concentratin of N




M
M
−
1 +
2
nsN
nsN
(9)



Pmix − PM Sum = 2 concentratin of N

(10)

The concentration of N could be directly obtained by
dividing Eq. (10) by 2.
For determination the concentration of the second component M, the same procedures should be repeated butwith using the normalized spectrum of M (ns M) as divisor
in all the previous steps instead of (ns N) or using Amplitude Difference method [24].
After estimation of the first component using ICE
method, the second drug in the binary mixture could bealso determined via other independent zero order recovering methods including Zero Order Extraction and Unity
Subtraction method [25].

for resolution of zero order spectrum of the second component in the binary mixture after estimation of the first
via applying successive spectral manipulation steps. US
method starts by multiplying the obtained N concentrationin the ICE to extract its zero-order spectrum D° N in thebinary mixture as mentioned in Eq. (11). The extracted D°
N is used as a divisor for the whole spectrum of the binary
mixture as follows.

D◦ M
D◦ N
D◦ mix
=
+
D◦ N
D◦ N
D◦ N

(13)

D◦ M
D◦ mix
= ◦
+1
◦
D N
D N

(14)

By subtracting the constant value 1 from the obtained◦
ratio spectrum DD◦mix
N and then multiplying by the samedivisor used (D° N), the original zero order spectrum ofM (D° M) will be resolved and directly determined at its
λmax as summarized in brief.

D◦ M
D◦ mix
−
1
=
+1−1
D◦ N
D◦ N

(15)

D◦ M
D◦ mix−1=
× D◦ N
D◦ N
D◦ N

(16)

D◦ mix− 1 = D◦ M
D◦ N

(17)

Zero order extraction method (Z°E)

The method aims at obtaining the original zero order spectrum of the compound under study which is considered
to be a unique fingerprint for each compound and hence
enabling its estimation at the wavelength of maximum
absorption (λmax). The method could be easily performed
through two simple steps. The first step is multiplying theobtained concentration of N, via ICE method, by its normalized spectrum to recover the whole zero order spectrum of N (D° N) in the binary mixture [21, 23]. The (D°N) is then subtracted from the total spectrum of the binary
mixture (D° mix) to finally extract the zero-order spectrumof M (D° M) actually found in the mixture. The concentration of M is determined through a correlation between theabsorbance of different concentration at its λmax and therespective concentrations. ZE method is an independent
method and could only be performed after quantificationof the first component of the mixture as summarized in thefollowing two steps.

Experimental
Apparatus

Shimadzu’s UV–Vis 1601 PC spectrophotometer(Tokyo, Japan) was used for the spectral analysis. Zeroorder spectra of the prepared solution were recorded
over a wavelength range of 200 to 400 nm.
TLC chromatography was performed on precoated
TLC plates (20 × 20 cm, 0.22 mm thickness) using silica
gel 60 GF254 from Merck (Darmstadt, Germany). For

The Concentration multiplication step: Concentration of N × ns N = D◦ N
The Absorption Subtraction step: D◦ mix − D◦ N = D◦ M

(12)

Unity subtraction method (US)The unity subtraction method (US) also known as unified constant subtraction is one of fingerprint resolving
technique previously reported [25]. It could be adopted(11)

data collection, a Linomat 5 auto sampler and scannerfrom Camag (Muttenz, Switzerland) was utilized. To
apply the sample, a Hamilton 100 μL syringe (Bonaduz,
Switzerland) was used. The scanning mode was absorbance, and the slit dimension was 3 mm × 0.45 mm witha scanning speed of 20 mm ­s−1.

Mohamed et al. BMC Chemistry(2024) 18:160

Page 5 of 16

Materials and reagents

Chemi-Pharm Pharmaceutical Company (Cairo, Egypt)
generously provided LCF, while Memphis Pharmaceutical Company (Cairo, Egypt) generously provided
CPM. For CPM and LCF, the purity was reported to be(99.89%) and (99.92%), respectively. Ethanol was of analytical grade and was purchased from Sigma-Aldrich(Darmstadt, Germany). El-Nasr Pharmaceutical Company (Cairo, Egypt) supplied analytical grades of toluene and ammonia (25%, v/v).
Lupituss-CPM® syrup (Lot number: M181259), containing 20 mg LCF and 4 mg CPM per 5 mL, was purchased from Lupin Ltd. (Mumbai, India).
Standard solutions and laboratory prepared mixtures

In order to prepare separate stock standard solutions of CPM and LCF (1 mg ­mL−1), ethanol was used
as a solvent. Working solutions were then prepared byappropriately diluting each stock solution with ethanol to get 0.1 mg ­mL−1 of each drug concentration.
Transferring aliquots from the drugs working solutions(0.1 mg ­mL−1), mixing them thoroughly, and filtering
them before adding ethanol to volume allowed to create
mixtures at various ratios for CPM and LCF. For spectrophotometric methods, the final ratios of both drugs
were (1:1, 1:2, 1:3, 1:5, 2:1, 3:1, 4:1 and 5:1, CPM: LCF,
respectively).

Spectroscopy procedures
Scanning of the zero order spectra

The spectrum of drugs under study using concentrationof 10 μg ­mL−1 for both were separately scanned within
200–400 nm wavelength range and intervals of 0.1 nm
intervals. The scanned spectra were recorded and storedwith the aid of the spectrophotometer software as displayed in Fig. 2. The spectra of both drugs were overlaid
and a sever overlap was observed with along the whole
wavelength range with no drug extended over the otherhindering their direct determination. New smart spectrophotometric method based on simple mathematical
approaches could be simply used for resolving tangled
spectra.
Preparation of calibration set and preparation of normalized
spectrum of LCF

Seven different concentrations were accurately preparedby transferring different aliquots equivalent to 30–300 μgof both CPM and LCF from their corresponding working solutions into two sets of 10-mL volumetric flasks.
The volume was completed to the mark using ethanol asa solvent. The spectra of different concentrations of bothdrugs were scanned within the 200–400 nm range using
the same solvent as the blank and saved using the spectrophotometer software. For the normalized spectrumof LCF; the stored spectra of different concentrations of

2.101
2.000

Abs.

1.500

1.000

0.500

0.000
200.00250.00Fig. 2 Zero order spectra of 10 μg ­mL−1 of CPM (―) and LCF (……)

300.00nm.

350.00400.00Mohamed et al. BMC Chemistry(2024) 18:160

LCF were recalled and separately divided by the respective concentration and an average spectrum (1 μg ­mL−1)
was recorded and stored.

Construction of calibration graphs
Independent concentration extraction (ICE) method

Solutions of different concentrations of pure CPM within
2–30 μg ­mL−1 were separately divided by the normalized
spectrum of LCF. The amplitude difference and summation at 263 nm and 300 nm were calculated for eachconcentration. A calibration curve was constructed relating the amplitude difference values against the amplitude summation values and the regression equation wascomputed.
Zero order extraction (Z°E) and unity subtraction methods(US)

Two separate calibration curves were constructed; thefirst one represented a correlation between the absorbance values of different LCF standard solutions at its
λmax 250.6 nm against the corresponding concentration. The second calibration graph is constructed
between the absorbance values at λmax 261.6 nm fordifferent standard solutions of CPM and corresponding concentrations. The respective regression equationfor each of CPM and LCF was computed to be used
for calculating unknown concentrations for both drugs
respectively.

Page 6 of 16

TLC procedures
Optimization of chromatographic conditionsfor HPTLC‑densitometry

The analysis was performed on pre-coated 20 × 10 cm
TLC aluminium silica gel 60 GF254 plates. An amount of
10 μL samples were applied to the plates at 3 mm band
width and at intermittent spaces of 10 mm between
bands. The injection was performed at 1 cm distancefrom the bottom edge. The mobile phase was used to presaturate the chromatographic chamber for 20 min before
developing it in an ascending fashion with toluene: As
the mobile phase, ethanol: acetone: ammonia (3:3:3:1, by
volume) was used. Separate aliquots of both drugs’ working solutions were applied to the TLC plates in triplicate,
which were then air dried at room temperature before
being scanned at 230 nm with a CAMAG TLC scanner.
Construction of calibration graphs

To obtain final concentrations of 0.3–4.0 μg/spot for
CPM and 0.8–10 μg/spot for LCF, aliquots from theworking standard solutions of CPM and LCF were accurately measured and separately applied to TLC plates
in triplicates. An aliquot of 10 μL of each solution wasapplied to pre-washed activated plates, and the plates
were developed with a mobile phase of toluene, ethanol, acetone, and ammonia acid (3:3:3:1 by volume). The
appropriate drug concentration was then plotted against
the peak area to generate a regression equation.
Analysis of laboratory prepared mixtures

Analysis of laboratory prepared mixtures
To apply the ICE method, The ­D0 of the synthetic binary
mixtures previously prepared was separately divided by
the normalized spectrum of LCF. A new ratio spectrum
was obtained for each mixture where the amplitude values at 263 nm and 300 nm were recorded. The amplitude difference (ΔP mix) and amplitude summation (∑Pmix) were calculated for each mixture. The amplitudedifference values were further manipulated to computethe unknown amplitude summation value (­PCPM sum)of standard CPM using the corresponding regression
equation. The ­(PCPM sum) is then subtracted from thetotal (∑P mix) to obtain a constant value resembling
the concentration of LCF in the mixture multiplied by
2 (2 × ­CLCF). The constant is then divided by 2 to obtainthe concentration of LCF. The same steps could be
repeated to obtain the concentration of CPM but using
its normalized spectrum instead of that of LCF as a divisor, but for simplification ICE was coupled with Z°E and
US to estimate the concentration of CPM at its λmax
using the corresponding regression equation.

Solutions containing the above-mentioned ratios of CPM
and LCF were placed into 10 mL volumetric flasks, andthe volume was then completed with ethanol. The solutions were then analyzed using the chromatographic procedures previously described and the obtained regression
equations were used to compute the % found for eachdrug.
In a 100-mL volumetric flask, 25 mL of Lupituss-CPM®
Syrup, equivalent to 20 mg of CPM and 100 mg of LCF,
were dissolved in 25 mL of ethanol to obtain concentrations of 0.2 and 1.0 mg ­mL−1 for CPM and LCF, respectively. Different concentrations were analysed in themanner described under calibration curve construction for both techniques, and the concentrations of bothdrugs were calculated.
Analysis of pharmaceutical formulations

Results and discussion
Extraction approach

The overlaid spectra of CPM and LCF exhibited severe
overlap along the whole wavelength range selected forMohamed et al. BMC Chemistry(2024) 18:160

measurement. No zero crossing points or extension of
one drug over the other were displayed hindering thedirect estimation of the drugs. A new spectrophotometric platform based on extraction methodology was suggested for the resolution of CPM and LCF and binary
mixture. The technique involved three different methods.
The first one is the independent concentration extraction (ICE) method could directly extract the concentration of both components consequently with no need forany other complementary method or any specific prerequisite such as presence of isosbestic point, dual wavelength, or extension of one component over the other.
The only prerequisite to execute the method is preparing and recording the normalized spectrum of the drugof interest. The other two methods namely Zero Order
Extraction (Z°E) and Unity Subtraction (US) methods are
dependent methods used for determination of the second component following few simple steps but need tobe coupled with other methods for determination of thefirst drug.
The three proposed methods lie under the umbrellaof spectrophotometric extracting technique where they
extract important hidden features from the overlapped
spectra of CPM and LCF. The ICE directly extracts theconcentration of the main or first component using its
normalized spectrum as a divisor and upon its couplingwith either Z°E or US methods, the whole zero order

Page 7 of 16

spectrum of both components could be extracted with all
their inherent features and peaks enabling their accurate
estimation at their λmax. The order spectrum of any component is also considered to be a fingerprint and plays arole in determination of the purity index.
Independent concentration extraction (ICE)ICE starts by the dividing the binary mixture either synthetically prepared in the laboratory or in tablet dosage
form by the stored normalized spectrum of LCF where a
new ratio spectrum (CPM + LCF)/ns LCF that could also
be expressed as (CPM/ LCF + concentration of LCF) as
displayed in Fig. 3. As shown in Fig. 3, three normalized
spectra appear. The first one (Fig. 3a) represents a constant value obtained by dividing the original spectrumof LCF (10 µg ­mL−1) by the reference divisor spectrum(LCF, 1.0 µg ­mL−1) which was represented as a straightline parallel to the origin. The other 2 normalized spectra
represent the normalized spectra of CPM alone (Fig. 3b)
and CPM/LCF mixture (Fig. 3c) at the same concentrations (10 µg ­mL−1).
Two wavelengths with significantly different amplitudes were selected carefully on the above ratio spectra(263 nm and 300 nm). The amplitude at the chosen wavelengths was subtracted one time to get the amplitudedifference (ΔP) and added to get the summed amplitude(∑P) another time. The ΔP was further manipulated toFig. 3 UV/VIS normalized spectra of LCF (a; 10 µg ­mL−1), CPM (b; 10 µg ­mL−1) and binary mixture of LCF/CPM (c; 10 µg ­mL−1, each). The
normalization method was achieved by dividing the measured spectra by that of a reference of LCF (concentration 1 µg ­mL−1)

Mohamed et al. BMC Chemistry(2024) 18:160

calculate the summed of pure LCF from the previously
computed corresponding regression equation amplitude­(PLCF Sum). Upon subtracting ­PLCF Sum from the total∑P, the result is a constant representing double the concentration of LCF. Hence, the concentration of LCF in the
mixture could be obtained by dividing the constant by 2.
The same steps could be followed for direct determination of CPM concentration using its normalized spectrum as the divisor instead. To avoid repetition and to
display the extraction technique, which is the main aim,the ICE method was only used for determination of LCF
and coupled with other extracting methods for determination of CPM.
Zero order extraction method (Z°E)

The Zero Order Extraction method could also be applied
for determination of CPM concentration via extracting
its zero order absorption spectra. The concentration of
LCF, obtained using Independent Concentration Extraction, was multiplied by its normalized spectra (ns LCF) to
get the zero-absorption spectrum of LCF (D°LCF) originally present the binary mixture. Finally, the zero-orderabsorption spectrum (CPM) could be easily extracted bydirect subtraction of the D°LCF from the total absorption
spectrum of the binary mixture. The absorption value atits λmax (261.6 nm) was recorded and used to calculate
the concentration using the corresponding regression
equation.
Unity subtraction (US) method

Another facile approach to determine the concentration of CPM is the Unity subtraction method in which
the exact (D° LCF) obtained via applying ICE method, as
detailed under (3.1), was used as a divisor. Upon dividing
the zero-order spectrum of the binary mixture by the (D°LCF) having the same concentration of LCF present the◦
mixture, a ratio spectrum was obtained ( DD◦CPM
LCF + 1). To
get the zero-order spectrum of CPM, subtract number(1) from the above ratio spectrum and then multiply the◦
resulted spectrum ( DD◦CPM
LCF ) by the used divisor to finally◦obtain D CPM.
TLC‑densitometric method

The proposed method was based on the difference in Rf
values [26] between CPM and LCF, which results fromdifferences in their polarities and migration rates on silica
plates. To achieve the best separation of the two drugs,
different solvent systems were used to optimise the chromatographic conditions. In terms of mobile phase selection and optimization, efforts had been made to achieve
an eco-friendly solvent system without sacrificing theanalytical efficiency.

Page 8 of 16

Finding the best solvent system is usually the most difficult part of developing a TLC method, especially if themobile phase has never been reported before, as in ourcase. Although starting with a large proportion of nonpolar solvent, such as chloroform, hexane, or benzene,
is a common method for achieving good separation.
Because of their well-known environmental hazards,
these solvents were excluded from our trials. Several
trials were conducted using various solvent systems,
including ethanol, acetone, and ethyl acetate in various
ratios (2:4:4, 6:2:2, and 4:3:3 by volume), but no separation was obtained, even after modifying the pH with formic acid, acetic acid, or ammonia. Other systems were
tested, including toluene: ethanol: acetone in various
ratios (2:2:6, 4:2:4, 3:3:4), with the toluene: ethanol: acetone (3:3:4 by volume) system demonstrating good separation but with tailing of the two drugs. As a result, we
experimented with different pH levels by adding either
ammonia or glacial acetic acid to the developing mixture,finding that basic pH reduces CPM tailing and improves
LCF peak shape. So, the amount of ammonia was optimized to get a final composition of toluene: ethanol: acetone: ammonia in a ratio (3:3:3:1 by volume). At 230 nm,
densitometric detection was carried out and Rf values
were 0.37 and 0.78 for CPM and LCF, respectively Fig. 4.
Method validation

Spectrophotometric method (extraction technique,which included ICE, Z°E, US) and HPTLC-densitometric methods were validated according to the recent ICH
guidelines [27, 28] to evaluate linearity, sensitivity, specificity, accuracy and precision.
The linearity ranges for both drugs were applied within
3.0–30.0 μg ­mL−1 for the proposed spectrophotometricmethod under the above-described experimental conditions. The validation parameters were summarised inTable 1. For HPTLC method, linear calibration curves
between peak areas and corresponding drug concentrations were obtained within concentration ranges of
0.3–4.0 μg/spot and 0.8–10.0 μg/spot for CPM and LCF,
respectively (Fig. 4). Table 1 shows the regression equations and regression parameters that were obtained.
The proposed HPTLC method’s sensitivity was evaluated and stated based on LOD (3.3 SD/S) and LOQ (10SD/S), where SD is the standard deviation of multiple
blank samples and S is the slope of the drug calibration curve, and the results are shown in Table 1. Meanwhile, for the novel ICE technique, LOQ was determinedaccording to the ICH recent guidelines [29] based on visual evaluation. The concentrations of LCF and CPM wereprepared in dilutions at lower concentration s below thelower concentration range. The ICE method was used toMohamed et al. BMC Chemistry(2024) 18:160

Page 9 of 16

Fig. 4 Two-dimensional (A) and three-dimensional (B) HPTLC densitogram of (4.0 μg/spot) of CPM and LCF at 230 nm, detection wavelengthMohamed et al. BMC Chemistry(2024) 18:160

Page 10 of 16

Table 1 Analytical performance data for the determination of the studied drugs by the proposed spectrophotometric and HPTLCdensitometric methods
Parameters

Linearity range

ICE

Z°E/US

TLC-densitometric

CPM

LCF

CPM

LCF

3.0–30.0 μg ­mL−1

3.0–30.0 μg ­mL−1

0.3–4.0 μg/spot

0.8–10.0 μg/spot
3078.49 ± 20.19

Regression equation*

Slope ± SD

1.2393 ± 0.00003

0.0397 ± 0.00019

3604.31 ± 13.95

Intercept ± confidence ­interval*

0.0017 ± 0.0006

0.0076 ± 0.003

743.28 ± 26.334

122.46 ± 19.293

Correlation Coefficient (r)

0.999

0.999

0.999

0.999

LOD

0.13 μg ­mL−1

0.66 μg ­mL−1

0.03

0.13

LOQ

−1

2.00 μg ­mL−1

0.09 μg/spot

0.40 μg/spot

0.40 μg ­mL

x ± t √sn where x is the mean value, and s is the standard deviation at n = 8 and the statistic value of t is 2.365 at 95% confidence interval with 7 degrees of freedom

Table 2 Application of the proposed spectrophotometric and HPTLC-densitometric methods for the determination of the studied
drugs in pure form
Drug

CPM

UV–Vis SpectrophotometryTLC-densitometry*

Reported method [16]Conc. added(μg ­mL−1)

Found (μg %Recovery­mL−1)

Conc. added(μg/spot)Found(μg/spot)%Recovery

Taken (μg­mL−1)

Found* (μg %Recovery­mL−1)

6

5.96

99.33

0.3

0.30

102.4010

10.04

100.448

8.13

101.63

0.7

0.69

98.37

20

19.91

99.56

12

11.98

99.83

1

1.01

100.9130

30.04

100.1516

16.09

100.56

2

1.99

99.98

22

21.91

99.59

3

2.98

99.48

28

28.31

101.11

4

4.01

100.28

Mean% ± SD*

100.34 ± 0.90

100.24 ± 1.36

100.05 ± 0.45

t-test0.51 (2.36)

LCF

6

6.04

100.67

0.8

0.79

98.56

10

9.86

98.63

8

7.93

99.13

2

2.01

100.2520

20.27

101.36

12

12.26

102.1744.00

100.11

30

29.86

99.55

16

15.89

99.31

6

5.97

99.46

22

22.18

100.8288.08

101.05

28

27.87

99.54

10

9.95

99.50

Mean% ± SD
t-test0.22 (2.36)

100.27 ± 1.16
0.40(2.36)

99.82 ± 0.85

99.85 ± 1.39

0.04 (2.36)*

Comparison method involved HPLC determination of CPM and LCF using C18 column with mobile phase of buffer (pH 6.5) and acetonitrile (50:50, % v/v) as isocratic
mobile phase, run time 8 min, flow rate 1 mL/min,linearity ranges 2–6 μg m
­ L−1 and 10–30 μg ­mL−1 for CPM and LCF respectively and UV detection at 227 nm

calculate each drug’s concentration to the minimum levelat which the analyte was resolved and quantified reliably.
Table 1 shows also the LOQ as obtained practically forboth drugs using the proposed novel ICE technique.
The proposed methods’ accuracy was investigatedby comparing the results to the reported method [16].
According to Statistical analysis of the data [30], there
was no significant difference in accuracy between them
(Table 2). Table 3 shows the results of tests on repeatability and intermediate precision. The RSD% values were
found to be less than 2 units, confirming the proposed
method’s high precision.

The selectivity of the proposed ICE method wasevaluated by the analysis of different laboratory prepared mixtures of CPM and LCF containing different
ratios within the linearity range and good results wereobtained and summarized in Table 4. The proposed
method’s validity was further evaluated by using thestandard addition technique and calculating the concentrations of standard added. Table 5 summarizes theacceptable results that were obtained.
Regarding the developed HPTLC method, the capacity factor (Kʹ), number of theoretical plates (N), resolution (Rs), tailing factor, and selectivity factor (α)

*

4.0

0.8

5.0

10.0

30.0

5.0

15.0

30.0

0.3

2.0

5.0

15.0

a

b

100.21 ± 0.55

99.98 ± 0.37

100.17 ± 0.47

100.30 ± 0.51

100.26 ± 0.55

100.02 ± 0.41

0.55

0.37

0.47

0.50

0.55

0.42

100.18 ± 0.73

99.83 ± 1.52

101.59 ± 0.54

99.96 ± 1.26

99.41 ± 0.97

101.87 ± 0.23

Mean ± ­SD*

Taken (μg/ spot)

Taken (μg m­ L−1)

0.73

1.52

0.53

1.26

0.98

0.23

Precision(RSD%)

Mean ± ­SD*

UV–Vis method

HPTLC-densitometry

UV–Vis method
Precision(RSD%)

HPTLC- Densitometric

Intra-daya

MethodAverage recovery results of three determinations; within the day; within three consecutive days using the proposed UV–Vis and HPTLC methods

LCF

CPM

Drug

100.33 ± 0.09

99.65 ± 0.25

100.07 ± 0.41

100.42 ± 0.05

100.28 ± 0.04

100.13 ± 0.12

Mean ± ­SD*

UV–Vis method

Inter-dayb

0.08

0.24

0.41

0.05

0.04

0.11

Precision(RSD%)

Table 3 Precision data for the determination of the studied drugs by the proposed spectrophotometric and HPTLC-densitometric methods

101.82 ± 1.07

100.74 ± 1. 12

1.05

1.11

1.33

0.85

98.85 ± 0.84
101.29 ± 1.35

1.60
1.02

99.65 ± 1.02

Precision(RSD%)
101.97 ± 1.63

Mean ± ­SD*

HPTLC-densitometricmethodMohamed et al. BMC Chemistry(2024) 18:160
Page 11 of 16

Mohamed et al. BMC Chemistry(2024) 18:160

Page 12 of 16

Table 4 Determination of CPM with LCF in laboratory prepared mixtures by the proposed spectrophotometric method
Ratio
CPM:LCFUV–Vis spectrophotometric method

TLC method

Laboratory preparedmixtures (μg ­mL−1)

Recovery%*Laboratory preparedmixtures (μg ­mL−1)

Recovery%*

CPM

CPM

CPM

CPM

LCF

LCF

LCF

LCF

1:2

5

10

98.12

102.08

4

8

99.15

98.12

1:1

20

20

101.98

98.31

2

2

100.3

101.41

3:1

30

10

101.71

98.96

3

1

99.66

99.55

2:1

20

10

100.66101.18

4

2

100.8

98.68

5:1

25

5

101.09

99.23

4

0.8

100.71

99.74

4:1

20

5

102.31100.48

4

1

98.97

100.2
101.11

1:5

5

25

100.05100.93

2

10

99.21

1:3

5

15

99.87

99.45

1

3

100.2399.02

100.72 ± 1.37

100.07 ± 1.29

Mean% ± SD

99.88 ± 0.73

99.73 ± 1.15

Mean% ± SD*

Average of three separate determinations

Table 5 Application of standard addition technique for the determination of CPM in combined Lupituss CPM® syrup formulations
with LCF by the proposed TLC- densitometric and spectrophotometric methods
CPM
Mean% ± RSD

LCF
Mean% ± RSD

Standard addition
Taken
μg ­mL−1

Added
μg ­mL−1

Recovery % of added

5.0
10.0
15.0

102.32100.15
99.56

Standard addition
Taken
μg ­mL−1

Added
μg ­mL−1

Recovery % of
added

10.0

5.0
10.0
15.0

101.1899.47
99.84

Spectrophotometric method
99.08 ± 0.75

10.0

Mean ± RSD%

100.31 ± 0.64

100.68 ± 1.45

Mean ± RSD%

100.16 ± 0.90

HPTLC-densitometry method
99.55 ± 1.93

1.0

Mean % ± RSD

0.3
2.0
3.0

101.33
98.89
100.4999.68 ± 0.84

100.24 ± 1.24

2.0

0.8
6.0
8.0

99.33
98.32
101.75

Mean % ± RSD

99.82 ± 1.76

were calculated to ensure system performance basedon United States Pharmacopeia (USP) guidelines [31]before or during the analysis, and the system wasfound to be suitable (Table 6).

Table 6 Parameters of system suitability test of the HPTLCdensitometric method

Application to pharmaceutical formulations

The proposed methods were used to determine CPM
and LCF in Lupituss-CPM® syrup. There was no interference from additives or excipients observed in the
results. As shown in Table 7, the calculated t-test values
are less than the tabulated ones, indicating no significant difference between the reported [16] and proposed
methods, confirming accuracy and precision at 95%
confidence limit.

Parameter

CPM

LCF

Reference value

Retardation factor ­(Rf)

0.37

0.78

Less than 1

Capacity factor (K)

1.70

0.28

The higher the value longer
the retention factor

Resolution ­(Rs)

2.87

Tailing factor

0.92

Selectivity factor (α)

6.07

>1
0.8

= 1 for typical symmetric peak>1

In conclusion, the ICE method employs an innovative extraction strategy that allows for independent
determination of the target compound’s concentration,

Mohamed et al. BMC Chemistry(2024) 18:160

Page 13 of 16

Table 7 Assay results for the determination of CPM in combined dosage forms with LCF by the proposed spectrophotometric, HPTLCdensitometric and reference [16] methods
Dosage form

Lupituss CPM® syrup

Spectrophotometric method

TLC- Densitometric method

Reported method [16]Conc.
taken (μg­mL−1)

Conc.
taken (μg/
spot)

Conc.
taken (μg­mL−1)% Found% Found% Found

CPM

LCF

CPM

LCF

CPM

LCF

CPM

LCF

CPM

LCF

CPM

LCF

3.0

15.0

98.89

101.020.3

1.5

101.77100.442.0

10.0

101.38

99.58

4.0

20.0

99.92

100.16

1.0

5.0

98.25

99.83

5.0

25.0

98.24

99.56

5.0

25.0

98.45

99.76

2.0

10.0

98.63

98.78

6.0

30.0

99.83

100.7899.82 ± 1.57

99.97 ± 0.71

Mean ± SD

99.08 ± 0.75

100.31 ± 0.64

99.55 ± 1.93

99.68 ± 0.84

t-test0.73 (2.776)

0.62 (2.776)

0.19 (2.776)

0.46 (2.776)

overcoming matrix interferences and providing accurate
measurements. By effectively separating the target compound from the complex matrix, ICE eliminates potential
biases and enhances the robustness of the analysis. The
method can be generalized to other mixtures in different overlay scenarios as proved by its advantages over theother classical methods.
Evaluating the sustainability of the proposed method

The concept of green analytical chemistry is critical forthe environment because it is urgent to achieve a highdegree of greenness by reducing or eliminating hazards
associated with chemical processes. The assessment of
the greenness of analytical methods has recently gained
motivation using various recent tools.
The green analytical procedure index (GAPI) is a recent
tool for assessing the green character of the entire analytical procedure, from sample collection to sample preservation, transport, preparation, and finally determination
in a pictogram composed of 15 zones [17]. A chart comprising the enumerated 15 zones is illustrated in supplementary materials Fig. S1 to clarify the assessment. GAPI
evaluates the ecological impacts using a pictogram withfive colored pentagrams representing each step of a procedure; there are three levels of color; green for low, yellow for medium, and red for high environmental impact.
In general, the greater the number of steps involved in
the procedure, the lower the greenness because energy
consumption and waste volume will increase. For the
first sample collection step, which is the time lag between
sampling and determination. AGREE is another highly
cited metric for evaluation that uses a numeric score.
AGREE uses the same color code as GAPI, but employing
the twelve principles of green analytical chemistry [18].
The green assessment profiles for the proposed methods
compared to previously reported RP-HPLC method [16]on GAPI and AGREE are shown in Table 8. As revealed,

GAPI pictograms show the best ecological impact for,the proposed spectrophotometric approach, then the
proposed HPTLC, and the lowest is represented for thereference RP-HPLC method [16], as indicated by the
number of red zones compared to green and yellow color
codes. When AGREE pictograms are studied (Table 8;
Supplementary materials table S2), the proposed spectroscopic approach suggested having the highest greenness score. Although the proposed HPTLC method haveslightly better score than the reference HPLC method[16], however, in this situation we can clarify the consof AGREE assessment compared to GAPI. AGREE metric doesn’t consider the waste treatment and recycling
scoring compared to GAPI, where the proposed HPTLC
method can recycle the mobile phase for several chromatographic runs compared to HPLC technique. Thiscan be shown by the lower right pictogram color codefor GAPI pictograms, where only the proposed HPTLC
method can show green color for waste treatment compared to the proposed spectroscopic approach (yellow)
and the reference method (red). The assessment tools
indicate that the proposed methods can be better used
for routine analysis of the studied mixture without causing environmental harm.
White analytical chemistry (WAC) metric is a more
recent tool which was created to assess analytical procedures from a variety of aspects [19], including efficiency,
ecological impact, and cost-effectiveness. Numerous
approaches exist for determining the long-term viabilityof a project, each with their own advantages and disadvantages. However, one strategy that has shown constantsuccess is to employ several diverse strategies simultaneously. By combining the concepts of red, green, andblue, we may analyze the procedure’s sustainability andarrive at a white result. As the red color indicates, theanalysis was effective. Analytical data on red parameters
contains information such as range of use, detection andquantification limits, accuracy, and precision. The twelve

Mohamed et al. BMC Chemistry(2024) 18:160

Page 14 of 16

Table 8 Assessment of the proposed and reported methods for determination of CPM and LCF
MethodWhite analytical chemistry

GAPI

AGREE

Proposed HPTLC method

Proposed spectroscopic method

Reported HPLC method [16]principles of green analytical chemistry (GAC) address
the issues of reagent toxicity, reagent quantity, waste,energy use, and direct consequences. The blue represents input that relates to needs, requirements, and easeof operation. In a downloadable Excel spreadsheet, wegive a table with three columns (red, green, and blue) to
let you compare the long-term viability of two different
approaches to analysis using the WAC metric. Based on
the information provided, a score out of 100 is determined, which quantifies the whiteness of the analysis.
This chart shows the percentage of each color and theirmerged white result. Scanning enables us to learn andanalyze the effectiveness and whiteness of the analytical procedure being utilized. A high percentage of eachcolor, but especially white, should result from the mostefficient and sustainable analytical process.
A comparison of the published RP-HPLC [16] resultswith the proposed methods utilizing the WAC metric (Table 8; Supplementary materials document 2)
reveals inconsistencies in the three-color parameters.
The proposed spectrophotometric and HPTLC methods demonstrated superior performance comparedMohamed et al. BMC Chemistry(2024) 18:160

to the reported HPLC method in terms of measuring
red color, achieving scores of 105.0% and 110.0% versus 97.5%, respectively. This can be attributed to theirheightened sensitivity, as evidenced by their lower limits of detection and quantification. Similarly, for green
color, the proposed spectrophotometric and HPTLC
methods outperformed the reported HPLC method,achieving scores of 96.3% and 87.1% versus 78.3%,
respectively. This can be attributed to the reduced consumption of solvent, energy, and waste associated withthe proposed methods. Furthermore, in terms of blue
color, the proposed spectrophotometric and HPTLC
methods also outperformed the reported HPLC
method, achieving scores of 81.5% and 79.6% versus
75.6%, respectively. The proposed spectrophotometricand thin-layer chromatography (TLC) methods exhibit
whiteness scores of 94.2% and 92.2% respectively, indicating a higher level of environmental friendliness compared to the reported method with a score of 83.8% asshown in Table 8.
Finally, the two proposed analytical techniques havebeen established and validated for the aimed purpose.
The choice between the novel ICE/UV–Vis spectroscopy and HPTLC methods for the analysis of the drugs
under study depends on the specific requirements of
the analysis and availability of tools. Both techniques
have their advantages and limitations. UV spectroscopy
is widely used as a relatively simple and quick technique. Meanwhile, HPTLC is a separation techniquethat requires minimal sample preparation and is particularly useful for low concentrations of the analytes.
However, it’s worth noting that the ecological impact of
ICE tool is lower than that of the HPTLC methodology.
Sometimes it may be beneficial to use both techniquesin combination to complement each other’s strengths
and/or limitations.

Page 15 of 16

effort during analysis. Both techniques are simple, accurate, precise and ecofriendly. Although toluene had beenused in the developed HPTLC method, it was used atminor amounts without greatly affecting the method’s
greenness as showed by the greenness assessment, andhence rendering the developed HPTLC method of lower
greenness compared to the developed spectrophotometric method. In addition, both techniques are cost effective where they only require a spectrophotometer witha built-in software for spectral manipulation and a densitometer with no need for any sophisticated programs.
The suggested methods could be applied in any quality
control lab for checking and fast track the concentration
change and/or degradation of drugs under study during
any production stage, packaging and on shelves to assure
safety and effectiveness of the marketed dosage forms.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​024-​01260-w.
Supplementary Material 1.
Supplementary Material 2.
Author contributions
All authors contributed substantially to the body of work. Ekram H. Mohamed,
Hany A. Batakoushy, Adel Ehab Ibrahim, Zeinab Adel Nasr, Marwa M. Soliman,
Sona S. Barghash, Tahany F. Mohamed, and Fatma A. Fouad; All authors havecontributed equally to the body of this work concerning conceptualization,
investigation, methodology, validation, writing, review, and editing. Ekram H.
Mohamed and Fatma A. Fouad; Project administration.
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB).
Availability of data and materials
No datasets were generated or analysed during the current study.

Declarations

Conclusion
According to the obtained results, it could be concluded
that the suggested analytical techniques demonstrated
selective and sensitive detection for both Chlorpheniramine maleate and Levocloperastine fendizoate either
in their pure form or in combination. The spectrophotometric platform covered a wider linearity range while
the chromatographic one showed more sensitivity. It is
worth mentioning that the proposed ICE spectrophotometric method is feasible to be adopted for the analysis
of complex binary and multi-component mixtures. In
addition, the newly adopted methods have the privilegeof being based on the extraction of the parent zero-order
spectra of the cited drugs and measuring the concentrations at their λmax with maximum sensitivity and less

Ethics approval and consent to participate
Not applicable. This research does not involve any humans or animals.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, The
British University in Egypt, El Sherouk City 11837, Egypt. 2 Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Menoufia University, Shebin Elkom 32511, Egypt. 3 Pharmaceutical Analytical Chemistry Department,
Faculty of Pharmacy, Port-Said University, Port Said 42511, Egypt. 4 Natural
and Medical Sciences Research Center, University of Nizwa, Birkat Al Mauz, P.O.
Box 33, Nizwa 616, Oman. 5 Pharmaceutical Analytical Chemistry Department,
Faculty of Pharmacy (Girls), Al-Azhar University, Cairo 11754, Egypt.

Mohamed et al. BMC Chemistry(2024) 18:160

Received: 25 February 2024 Accepted: 2 August 2024

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