(2024) 18:149
katamesh et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01254-8

BMC Chemistry

RESEARCH

Open AccessGreen micellar UPLC and complementaryeco‑friendly spectroscopic techniques
for simultaneous analysis of anti‑COVID drugs:
a comprehensive evaluation of greenness,blueness, and whitenessNoha S. katamesh1, Ahmed Emad F. Abbas2* , Michael K. Halim2, Mohamed A. Abdel‑Lateef3 andShimaa A. Mahmoud1

Abstract
The development of sustainable analytical methodologies that minimize hazards, waste generation, and energy
consumption has become crucial. This study introduces pioneering green‒blue-white approaches for the simultane‑
ous quantification of montelukast sodium (MLK) and fexofenadine hydrochloride (FEX) in combination formulations.
The first approach employs an ultra-performance liquid chromatographic method (UPLC) with a green micellar
mobile phase of 0.02 M sodium dodecyl sulfate and 10% 1-pentanol (65:35%). The method demonstrated excellent
resolution, peak symmetry, and a short analysis time, with retention times of 3.53 min for MLK and 1.67 min for FEX.
The MLK and FEX linearities were 1–260 and 1.2–312 μg/mL, respectively. The second approach involves comple‑
mentary built-in spectroscopic techniques (second derivative, third derivative, and ratio difference methods) using
water as a solvent, providing a green, simple, low-cost alternative in laboratories where expensive chromatographic
devices may not be readily available. The MLK and FEX linearities were 3–50 and 3–60 μg/mL, respectively. All meth‑ods were comprehensively validated and showed satisfactory results. The proposed methods demonstrated excellent
linearity ­(r2 ≥ 0.9990), accuracy (recovery 98.5–101.5%), and precision (RSD ≤ 2%) across wide concentration ranges.
A multifaceted evaluation was conducted to assess the environmental sustainability, real-world applicability, and eco‑nomic viability of the proposed methods in comparison with previously reported techniques. This comprehensive
assessment leveraged several state-of-the-art tools, including NEMI, ComplexGAPI, AGREE, ESA, BAGI, and RGB12. The
suggested approaches exhibited favorable quadrant profiles in the NEMI and ComplexGAPI assessments, coupledwith higher AGREE scores (0.90, 0.86) than reported (0.62, 0.74, 0.75, 0.69, 0.74, 0.74, and 0.75), in addition to higher
ESA score (88, 92) than reported (75, 84, 85, 79, 82, 82, and 83), collectively affirming their environmentally friendly
credentials. Moreover, we embraced the innovative notions of ’blueness’ and ’whiteness’ assessment by harnessing
the recently formulated BAGI and RGB12 algorithms. The higher BAGI score (90, 82.5) than reported (72.5, 70, 70, 67.5,
67.5, 67.5, and 72.5), confirmed the excellent real-world applicability of the proposed methods, while the notable*Correspondence:
Ahmed Emad F. Abbas
dr.ahmedeemad@gmail.com; ahmed.emad.pha@o6u.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
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katamesh et al. BMC Chemistry(2024) 18:149

Page 2 of 20

RGB12 indices (89.8, 88.1) than reported (67.8, 72.8, 71.5, 67.1, 73.7, 70.3, and 73.2), validated their cost-effectivenessand overall sustainability, contributing to an eco-friendly future for quality control processes.
Keywords Montelukast sodium, Fexofenadine hydrochloride, Green micellar UPLC, Eco-friendly spectroscopy,
Greenness, blueness and whiteness assessment

Introduction
Green analytical chemistry (GAC) and white analytical chemistry (WAC) represent transformative shifts inanalytical methodologies, advocating for practices thatminimize environmental impacts and enhance efficiency
throughout the analytical lifecycle [1–5]. GAC prioritizes
reducing sample preparation, decreasing energy usage,and eliminating toxic reagents to prevent pollution,
enabling waste recycling, and promoting safer chemical
production. Similarly, WAC focuses on efficiency, costeffectiveness, and practicality, guiding the maximizationof productivity with minimal resource consumption.
Pharmaceutical quantification, as a cornerstone ofanalytical endeavors, demands meticulous adherence tothese combined green-and-white analytical principles[6–8]. While traditional performance criteria such as
accuracy and sensitivity remain crucial, environmental
responsibility and judicious resource usage are equally
paramount [9]. Solvents such as acetonitrile and methanol, common in conventional methods, conflict with
GAC standards due to their appreciable toxicity. Additionally, instruments with prohibitive costs may undermine the practical objectives of WAC [10].
The significance of developing greener methods is
exemplified in the case of montelukast sodium (MLK)
and fexofenadine hydrochloride (FEX). Although the
combined use of MLK and FEX is widespread and important, existing analytical approaches for this drug combination have significant environmental drawbacks andlimitations. Therefore, there is a pressing need to develop
newer, greener analytical methods for this combination. MLK, as shown in Fig. 1a, is chemically generated
from 2-[1-[[(1R)-1-[3-[2-(7-chloroquinolin-2-yl) ethenyl] phenyl]-3- [hydroxypropan-2yl) phenyl] propyl]
sulfanylmethyl] cyclopropyl] acetic acid and sodium
salt [11]. In 2020, it was ranked as the fourteenth mostoften prescribed pharmaceutical in the U.S., with a totalof more than 31 million prescriptions as a result of its
significant role as a potential COVID-19 therapeutic[12]. FEX, as shown in Fig. 1b, is chemically converted
to 2-[4[1-Hydroxy-4-[4-(hydroxy-diphenyl methyl)-1-piperidyl] butyl] phenyl]-2-methyl propanoic acid [11]. In
2020, it was ranked as the 255th most commonly prescribed pharmaceutical in the U.S., with a total of morethan 1 million prescriptions as a result of the role of FEX
as a preventive drug for the treatment of severe acuterespiratory syndrome (SARS) because it acts as a potent
inhibitor of the main protease of COVID-19 [13]. The
concurrent administration of MLK and FEX in combination therapy regimens results in synergistic and complementary effects [14].
Various quantification techniques, including UV‒
Vis spectroscopy [15–20], HPLC [21–24], UPLC [25],
HPTLC [26], and LC–MS [27], have been established
for this drug combination. However, these methods arecostly and complex, use nongreen chemicals, and are
unsuitable for routine quality control analysis. UV-based
methods struggle with specificity issues, particularly
low sensitivity and the use of a harmful solvent, methanol, which is not compatible with green chemistry principles due to its potential toxicity and environmental
impact. HPLC techniques require run times exceeding 15
min per sample along with substantial volumes of toxic
organic solvents such as methanol and acetonitrile. The
existing HPTLC protocol, though faster, still relies on
unsustainable chlorinated mobile phases. The LC–MS
method faces barriers to widespread adoption due tohigh instrumentation costs and complexity. Comparedwith other methods, only one published UPLC method
offered some improvement in environmental sustainability; however, it still relies on chemicals that are not environmentally friendly, such as acetonitrile. This warrants
the development of alternate techniques that align moreclosely with GAC and WAC principles to balance efficacy
and eco-friendliness.
In this study, we aimed to achieve four core objectives. First, we developed a green UPLC method for thequantification of MLK and FEX using a green micellar
mobile phase that offers short analysis times, high selectivity, excellent resolution, peak symmetry, and optimallinearity and recovery. Second, develop an eco-friendly
complementary spectroscopic approach using water as
the solvent for the quantification of MLK and FEX thatprovides a green, simple, low-cost alternative in laboratories where expensive chromatographic devices may not
be readily available. Third, we comprehensively evaluated
the greenness of the proposed and reported methods
using established tools like the analytical eco-Scale (ESA)
tool, the analytical greenness metric (AGREE), the complex complementary green analytical procedure index(ComplexGAPI), and the national environmental method
index (NEMI). These ensure environmental benefitskatamesh et al. BMC Chemistry(2024) 18:149

Page 3 of 20

Fig. 1 Chemical structure of a montelukast sodium, b fexofenadine hydrochloride

compared to reported methods. Fourth, we employed
the recently proposed "blueness" and "whiteness" assessment tools, which have been gaining traction in evaluating the practical applicability and overall sustainabilityof analytical methods, respectively, for all proposed andreported methods, confirming their outstanding performance, dependability, and economic viability. The results
for the UPLC method and the three spectrophotometricmethods (second derivative, third derivative, and ratio
difference) are presented separately. For green assessment tools (AGREE, ESA, NEMI, and ComplexGAPI)and the BAGI tool, the UPLC method is reported, while
for spectrophotometric methods, we report one result
as there were no differences due to shared principles
and eco-friendly solvents and reagents. However, for the
RGB12 ’whiteness’ assessment, considering additional
validation parameters, the individual UPLC method
score is reported, while for spectrophotometric methods,
the average ’whiteness’ score across the three methods ispresented to account for differences in limits of detection(LOD). Finally, drawing attention to an exemplary modelfor environmentally friendly analysis without sacrificing
accuracy or practical performance exemplifies the commitment to a sustainable future in routine quality control
practice. In summary, we draw attention to an exemplarymodel for environmentally friendly analysis without sacrificing accuracy or practical performance exemplifies
the commitment to a sustainable future in routine quality
control practice.

Experimental
Reagents and materials

The solvents used were of HPLC grade, and all the compounds were of analytical reagent grade. Sodium dodecyl sulfate (SDS, 98.5%), methanol, sodium dihydrogen
phosphate, and 1-pentanol were acquired from Sigma‒
Aldrich (Steinheim, Germany). Ultrapure water was
obtained from a Milli-Q water purification system (Millipore, USA) and used throughout the analysis.

katamesh et al. BMC Chemistry(2024) 18:149

Samples
Reference standards• The reference standard of MLK (lot no. MK-0180513)
with a purity of 99.30% was kindly gifted by EGY
Pharm Pharmaceutical Company, Cairo, Egypt.
• The reference standard for FEX (lot no. F007040216,
99.89% purity) was generously provided by Memphis
Pharmaceutical Company, Cairo, Egypt.
The pharmaceutical formulation Montair-FX® tablets(Cipla Ltd., Mumbai, India) was purchased from a local
pharmacy. Per the label claim, each tablet was formulated
to contain 10 mg of MLK and 120 mg of FEX.
Pharmaceutical sample

Instrumentation• The micellar UPLC system consisted of an Agilent
Infinity 1290 model (Agilent Technologies, USA)equipped with a UV detector, a quaternary pump,
and a degasser unit DGU-20 (A5). Chromatographic
separation was performed on a Kinetex 1.7 μm
HILIC 100A column (100 × 2.1 mm, 1.7 μm particlesize) (Phenomenex, USA) maintained at 40 °C in acolumn oven.
• Absorbance measurements were made on a Shimadzu UV-1800 spectrophotometer (Shimadzu
Corp., Japan) with 1 cm path length quartz cuvettes.
The spectrophotometer was operated using in-built
UVProbe software version 2.21 with a scanningspeed of 2800 nm/min and bandwidth of 2 nm.
Standard solutions

Primary stock solutions of MLK and FEX (1 mg/mL for
UPLC, 100 μg/mL for spectrophotometry) were preparedby dissolving reference standards in ultrapure water.
Working standard solutions were freshly prepared byappropriate dilutions of stock solutions with ultrapure
water to yield the concentrations required for constructing calibration curves. The prepared solutions were stable
when refrigerated at 4 °C for 1 week.
Procedures
Micellar UPLC method

The micellar mobile phase consisted of 0.02 M SDS and
10% 1-pentanol (65:35%) buffered at pH 3.5 with 0.05 M
sodium dihydrogen phosphate. The mobile phase was filtered through a 0.22 μm membrane filter and degassed
ultrasonically prior to use. The flow rate, column temperature, and detection wavelength were maintained atPage 4 of 20

1 mL/min, 40 °C, and 230 nm, respectively. Under optimized conditions, the MLK and FEX peaks appearedat retention times of 1.67 and 3.54 min, respectively, as
shown in Fig. 2.
Spectroscopic methods

UV‒Vis spectra of standard MLK and FEX solutions
were acquired from 200–400 nm using water as a blank,
and the overlaid zero-order absorption spectra are presented in Fig. 3.
Construction of calibration curves
UPLC method

Appropriate volumes of standard drug solutions werediluted with the mobile phase to yield concentrations in
the range of 1–260 μg/mL for MLK and 1.2–312 μg/mL
for FEX. The solutions were subsequently examined byinjection into the chromatographic system. The calibration plots were generated by plotting the peak area versus
the corresponding concentration in μg/mL, after which
the relevant regression equation was calculated.
Spectroscopic methods

A series of 10 mL volumetric flasks were prepared withaliquots of standard MLK and FEX solutions ranging
from 3–50 μg/mL for MLK and 3–60 μg/mL for MLK
and FEX, respectively, and then filled to the required volume with water. The absorption spectra of the prepareddrug solutions were measured at 200–400 nm using
water as a blank.
Second derivative method (2D) The stored spectral data
were processed to obtain 2D spectra using built-in UVProbe software with a wavelength difference (Δλ) = 8 nm
and a scaling factor (SF) = 150. The peak amplitudes at 341
nm for MLK and 213.6 nm for FEX were measured. Calibration plots were generated by graphically representing
the peak amplitudes against their respective concentrations expressed in μg/mL, as illustrated in Fig. 4, and thepertinent regression equation was computed.
Third derivative method (3D) The 3D spectra wereobtained from the stored data using built-in UV-Probe
software with Δλ = 16 nm and SF = 6000. The peak amplitudes at 295, 336.4, and 346 nm for MLK and at 235 nm
for FEX were measured. Calibration plots were generated
by graphically representing the peak amplitudes against
their respective concentrations expressed in μg/mL, as
illustrated in Fig. 5, and the pertinent regression equation
was computed.
Ratio difference (RD) method The ratio spectra of MLK
were acquired by dividing the recorded MLK’s zero-orderkatamesh et al. BMC Chemistry(2024) 18:149

Page 5 of 20

Fig. 2 Micellar UPLC chromatogram demonstrating the separation of 4 μg/mL MLK and 48 μg/mL FEX from the laboratory-prepared mixture

absorption spectra by the FEX divisor spectrum (15 μg/mL). Similarly, the FEX ratio spectra were acquired by
dividing the recorded FEX zero-order absorption spectra by the MLK divisor spectrum (5 μg/mL). Calibration
curves were constructed by plotting the difference inamplitudes at 250 and 245 nm for MLK or 227 and 217
nm for FEX versus the corresponding concentrations, as
shown in Fig. S1, and the relevant regression equation was
calculated.
Analysis of laboratory‑prepared mixtures(a) UPLC method
Aliquots of MLK and FEX were added to 10 mL volumetric flasks, which were then filled with mobile
phase to yield different concentration ratios of thetwo drugs covering the linear range. The solutions
were mixed well and analyzed under optimized
UPLC conditions. The concentrations of MLK and
FEX were determined from the respective regression equations.

(b) Spectroscopic method
Aliquots of MLK and FEX were added to 10 mL volumetric flasks, which were then filled with water toyield different concentration ratios of the two drugs.
The absorption spectra of these laboratory mixtures
were measured and recorded as described earlier under linear conditions. The concentrations of
MLK and FEX were determined using the respective spectroscopic methods.
Pharmaceutical dosage form analysis

The proposed methods were applied to the quantitative
determination of MLK and FEX in their combined tablet formulation. Five Montair FX® tablets, each labeledwith 10 mg of MLK and 120 mg of FEX, were collectively
pulverized into a fine powder. An accurately weighedamount of the powdered tablets, equivalent to 10 mg
of MIK and 120 mg of FEX, was transferred into a 100mL volumetric flask. Approximately 50 mL of water
was introduced into the flask, and the mixture was subjected to sonication for 15 min to facilitate the extraction of the active pharmaceutical ingredients into thekatamesh et al. BMC Chemistry(2024) 18:149

Page 6 of 20

2.880
Fexofenadine hydrochloride (48 μg/mL)

Montelukast sodium (20 μg/mL)

Absorbance

2.000

Mixture (10 + 10 μg/mL)

1.000

0.000
200.00250.00300.00350.00400.00

Wavelength (nm)Fig. 3 Zero-order spectra of MLK and FEX and a mixture of MLK and FEX in distilled wateraqueous medium. The solution was then diluted to thefinal volume with water, thoroughly mixed, and filteredthrough a 0.45 μm membrane filter. Subsequent dilutions
with water yielded final concentrations of 10 μg/mL for
MLK and 120 μg/mL for FEX. Aliquots from the clear
filtrate were placed into 10 mL volumetric flasks andfurther diluted with water. The established procedures,as detailed previously, were meticulously followed, andregression equations were employed to determine theconcentrations of MLK and FEX in the tablet samples.

Results and discussion
The pursuit of analytical methodologies that harmonize
sustainability principles with high performance remains
a pressing challenge [8]. Consequently, the overarching
objective of this study was to devise an innovative andenvironmentally conscious approach for the simultaneous quantification of the anti-COVID pharmaceuticals
MLK and FEX. This was achieved through the strategic
integration of selective micellar UPLC and complementary spectroscopic techniques that leveraged highly sensitive detection and eco-friendly solvents. The strategic
integration of UPLC technology and eco-friendly micellar

liquid chromatography provides an optimal model forbalancing separation efficiency with green chemistry
aspirations [28]. Specifically, the UPLC column provides
exceptional peak resolution, sensitivity, and symmetry
within a drastically shortened run time of only 3.5 min,enhancing sample throughput. However, using that technique along with commonly used but environmentally
harmful liquid solvents like acetonitrile or methanol canthreaten sustainability efforts. Therefore, the introduction of a biocompatible micellar mobile phase containing
the surfactant SDS and pentanol modifier mitigates thislimitation, avoiding nearly all toxic solvent use. In liquid
chromatography (LC), the mobile phase typically consists of organic solvents, such as acetonitrile or methanol,
mixed with water. These organic solvents are often toxic
and environmentally harmful. Micellar liquid chromatography [29], on the other hand, uses an aqueous solution of a surfactant, such as SDS, as the mobile phase. In
aqueous solutions above a certain concentration (known
as the critical micelle concentration, CMC), SDS molecules self-assemble into colloidal aggregates called
micelles. These micelles have a hydrophobic core and ahydrophilic outer shell, which can mimic the behavior

katamesh et al. BMC Chemistry(2024) 18:149

1.044

Page 7 of 20

213.6 nm

FEX (15 μg mL-1 )MLK (15 μg mL-1 )0.500
341 nm0.000-0.500-0.866200.00250.00300.00350.00400.00

Wavelength (nm)Fig. 4 Second-order spectra of MLK and FEXof organic solvents in LC. The hydrophobic cores of themicelles can accommodate non-polar analytes, while
the polar outer shells can interact with polar analytes,
facilitating their partitioning and subsequent separation.
When an analyte is introduced into the micellar mobile
phase, it can partition between the aqueous phase andthe hydrophobic core of the micelles, similar to how it
would partition between the aqueous and organic phases
in RP-HPLC. This partitioning behavior enables the separation of analytes based on their hydrophobicity, just likein LC. This micellar UPLC approach thereby maximizes
chromatographic performance while manifesting thegreen analytical principles of benign-by-design chemicals
and waste minimization.
Likewise, transitioning to complementary spectroscopic techniques introduces a practical, low-cost
analytical alternative that further reduces solvent consumption by employing only water [30]. With accurate,
precise measurements rivaling complex assays, thesesustainable spectroscopic methods exemplify anothergreen chemistry model reconciling efficacy and environmentally responsible practices, which is particularly

valuable for modest laboratories lacking expensive
instrumentation. Moreover, the methods presented inthis study involved a multifaceted evaluation process
employing an array of complementary sustainability
assessment techniques. These comprehensive assessments aimed to scrutinize the green and white credentials of the proposed approaches. The results emerging
from these evaluations provided compelling evidenceof the remarkable alignment of the recommended strategies with the established parameters that define sustainable analytical chemistry practices.
UPLC method development and optimization

The most critical chromatographic parameters affecting
MLK and FEX separation were investigated and analyzed carefully to achieve satisfactory separation of thetwo drugs with symmetrical, sharp peak shapes within aminimal duration. The ICH guidelines [31] describe thecalculation of chromatographic performance in termsof the number of theoretical plates, resolution, and tailing factor, and the results are presented in Table S1.

katamesh et al. BMC Chemistry(2024) 18:149

Page 8 of 20

3.000
2.000

FEX (15 μg mL-1 )235 nmMLK (15 μg mL-1 )295 nm

336.4 nm

0.000

346 nm-2.000-3.948200.00250.00300.00350.00385.00

Wavelength (nm)Fig. 5 Third-order spectra of MLK and FEX

Selection of columns

To reduce solvent consumption and develop agreener method, we evaluated columns with shorter
lengths, smaller internal diameters, and smaller particle sizes. Several reversed-phase columns, including Kinetex® HILIC (100 × 2.1 mm, 1.7 μm), Kinetex
C18 (100 × 4.6 mm, 2.6 μm), Zorbax Eclipse XDBC18 (150 × 4.6 mm, 5 μm), and Monolithic RP-C18(100 × 4.6 mm), were tested for the optimal separationof MLK and FEX.
Through these trials, the Kinetex HILIC column
emerged as the most suitable choice, providing symmetrical peaks and the shortest analysis time of 3.5 min. The
superior performance of this column can be attributed toits favorable interactions with the chemical and physicalproperties of MLK and FEX [32].
Both MLK and FEX exhibit moderate polarity, with
topological polar surface areas (TPSA) of 73.25 Å2 and81 Å2, respectively, arising from the presence of hydroxyl
groups, a piperidine ring (in FEX), and other polar functional groups. The HILIC (Hydrophilic Interaction Liquid Chromatography) stationary phase is specificallydesigned to separate polar and ionizable compounds
through favorable interactions with these polar groups,

contributing to the efficient separation observed for MLK
and FEX.
Additionally, the pKa values of MLK (strongest basic
pKa of 3.49) and FEX (strongest basic pKa of 9.21) indicate their potential to exist as cations under certain pH
conditions. The HILIC mechanism involves electrostaticinteractions between the analytes and the stationary
phase, which can contribute to the separation based on
the ionization behavior of these compounds.
Furthermore, the relatively large molecular sizes of
MLK (608.17 g/mol) and FEX (501.67 g/mol), alongwith their complex structures, are accommodated by the
100 Å pore size of the Kinetex HILIC column, facilitating their retention and separation. Although the HILIC
mechanism primarily relies on polar interactions, thepresence of hydrophobic regions in MLK (logP 8.421)
and FEX (logP 2.939) can contribute to additional interactions with the stationary phase, potentially enhancing
their retention and selectivity.
In contrast, the other evaluated columns, such as
Kinetex C18, Zorbax Eclipse XDB-C18, and Monolithic
RP-C18, exhibited broader peaks, longer retention times,
and insufficient resolution, as shown in Fig S2. Thesecolumns may not have provided optimal separation

katamesh et al. BMC Chemistry(2024) 18:149

conditions for MLK and FEX based on their chemicaland physical properties.
Selection of detection wavelength

Various wavelengths, including 200, 210, 220, 230, 240,
250, and 260 nm, were tested. Based on spectral evaluation and the observation of a better baseline, 230 nm wasselected as the optimal detection wavelength, as shownin Fig S3. The choice of 230 nm is scientifically justified
by the fact that both MLK and FEX exhibit their maximum UV absorption around this wavelength due to thepresence of chromophoric groups in their structures.
This maximum absorption ensures the highest sensitivity during quantification, as it aligns with the optimalenergy level for the molecular interaction forces of thesecompounds.
Optimization of the mobile phase

Including improved safety, cost-effectiveness, and ecofriendliness. Micelles, colloidal aggregates of surfactants,
demonstrate excellent chromatographic efficiency. Additionally, micellar mobile phases enable greener analysis
by minimizing the use of toxic organic solvents.
To achieve optimal separation of MLK and FEX withina reasonable time frame, various mobile phase compositions were evaluated through trials. The impact of varying
the concentration of SDSs over the range of 0.01–0.04 M
on the retention times and separation of the drugs wasinvestigated (Table S1). The most effective resolution was
obtained with 0.02 M SDS.
Furthermore, different organic modifiers, including
methanol, acetonitrile, ethanol, n-propanol, n-butanol,and n-pentanol, were tested to enhance the separation(Fig S4). When ethanol and methanol were used, noseparation occurred for MLK and FEX, indicating theirincompatibility with the chemical properties of thesedrugs. The use of n-butanol, acetonitrile, or n-propanol
resulted in broad peaks with long retention times, suggesting suboptimal interactions with the analytes.
Additionally, the effect of varying the percentage of
1-pentanol from 5 to 20% was studied. It was foundthat incorporating 15% 1-pentanol in the mobile phaseyielded satisfactory resolution, symmetrical peaks,
and good peak clarity within a reasonable analysis time
(Table S1).
The selection of 0.02 M SDS and 15% 1-pentanol as the
optimal mobile phase composition is scientifically justified by the chemical and physical properties of MLK and
FEX. Both drugs possess moderate polarity, as indicatedby their topological polar surface areas (TPSA) of 73.25
Å2 for MLK and 81 Å2 for FEX, arising from the presence of hydroxyl groups, a piperidine ring (in FEX), andother polar functional groups. The micellar mobile phase,

Page 9 of 20

comprising SDS and the pentanol modifier, can effectively interact with these polar moieties, facilitating efficient separation.
Moreover, the pKa values of MLK (strongest basic pKaof 3.49) and FEX (strongest basic pKa of 9.21) indicate
their potential to exist as cations under certain pH conditions. The micellar mobile phase can engage in electrostatic interactions with the ionized forms of thesecompounds, further contributing to their separation
based on their ionization behavior.
Furthermore, the relatively large molecular sizes of
MLK (608.17 g/mol) and FEX (501.67 g/mol), alongwith their complex structures, are accommodated by
the micellar mobile phase, enabling their retention andseparation. Although the micellar mechanism primarily
relies on polar interactions, the presence of hydrophobic regions in MLK (logP 8.421) and FEX (logP 2.939)
can contribute to additional interactions with the micellar system, potentially enhancing their retention andselectivity.
Effects of pH and buffer concentration

The effect of pH on the chromatographic behavior of
MLK and FEX was investigated over the range of pH 3.0
to 4.5 using 0.05 M sodium dihydrogen phosphate buffer
in the mobile phase. A pH of 3.5 was found to provide
maximum theoretical plates and optimal peak symmetry
for both analytes.
Additionally, the impact of varying buffer concentrations was evaluated. Among the tested concentrations,0.05 M buffer led to well-resolved and sharp peak shapes,
as evident from the sample chromatogram (Table S1).
Consequently, a 0.05 M buffer at pH 3.5 was selected as
the final mobile phase condition.
The selection of pH 3.5 is scientifically justified by theionization behavior of MLK and FEX, as dictated bytheir pKa values. At this pH, MLK (strongest basic pKaof 3.49) is likely to exist in a partially ionized form, while
FEX (strongest basic pKa of 9.21) remains predominantly
neutral. The optimal peak shapes and resolution achievedat pH 3.5 suggest that this condition facilitates favorable
interactions between the analytes and the micellar mobile
phase, contributing to their efficient separation.
Effects of flow rate and column temperature

The flow rate was varied from 0.6 to 1.2 mL/min during
the optimization process. A flow rate of 1 mL/min was
found to be optimal, as it provided better peak shapes,
shorter retention times for both analytes and satisfactory
peak resolution.
Similarly, the column oven temperature was varied
from 30 °C to 50 °C. While higher temperatures increased
the theoretical plates, they also proportionately increased

katamesh et al. BMC Chemistry(2024) 18:149

peak tailing. To achieve the best peak shapes and resolution, a column temperature of 40 °C was maintained(Table S1).
The selection of a 1 mL/min flow rate and a column
temperature of 40 °C is scientifically justified based on
the principles of chromatographic separation and thephysical properties of MLK and FEX.
The flow rate directly influences the linear velocity ofthe mobile phase through the column, affecting the masstransfer kinetics and the equilibration time between thestationary and mobile phases. A flow rate of 1 mL/min
likely provides an optimal balance between efficient masstransfer and reasonable analysis time, contributing to theobserved peak shapes and resolution.
The column temperature, on the other hand, affects theviscosity of the mobile phase, the diffusion coefficients of
the analytes, and the interactions between the analytes
and the stationary phase. At 40 °C, the mobile phase viscosity and analyte diffusion rates are likely optimized,
facilitating efficient mass transfer and minimizing peaktailing. Additionally, the interactions between MLK, FEX,
and the micellar mobile phase may be optimal at thistemperature, contributing to the observed peak shapes
and resolution.
Spectrophotometric methods

The overlaid UV absorption spectra of MLK and FEX(Fig. 3) showed a severe overlap, making direct simultaneous estimation difficult. Hence, advanced spectroscopic
techniques were developed for their quantification. Threesimple, economical, and accurate derivative (2D, and 3D)
and ratio difference (RD) spectrophotometric methods
were developed.
Development and optimization

Selection of solvent The absorption intensity and solubility of the MLK and FEX standards were studied in distilledwater, ethanol, acetonitrile, methanol, and aqueous buffers of varying pH, such as 0.1 M HCl, pH 8 borate buffer,
0.1 M NaOH, and pH 4 acetate buffer. Distilled water gave
the maximum absorption intensity and solubility for bothdrugs and was selected as a blank/solvent for eco-friendly
analysis.
Delta lambda and scaling factor for the methods For
derivative spectrophotometry, the derivative order, Δλ,and SF concentration were optimized to achieve sufficientsensitivity and selectivity at working concentrations. The
initial assessment involved calculating the first derivativeof the ratio spectra, but no clear peaks were identified.
Consequently, the second and third derivatives of the ratio
spectra were employed. For the 2D method, parameters of
Δλ = 8 nm and SF = 150 gave well-defined peaks with thePage 10 of 20best signal-to-noise ratio. Similarly, for 3D, Δλ = 16 nm
and SF = 6000 were optimal.
Selection of divisors Different concentrations of FEX andMLK standards were evaluated as divisors to determine
the optimal RD method. FEX at concentrations of 15 μg/
mL and MLK at 5 μg/mL gave the best recovery with thehighest sensitivity and reproducibility.
Method characteristics

Second derivative method This technique involves theconversion of a normal zero-order absorption spectrum
to a 2D spectrum [30]. This approach helps eliminate
spectral interference by abolishing background effects
and improving detection selectivity. In this method, thezero-order absorption spectra of FEX and MLK weretransformed into 2D signals. Then, each was determinedat a zero-crossing point where the other had no absorbance at this point. In the present work, 2D spectra of MLK
and FEX were obtained by UV-Probe software using
Δλ = 8 nm and SF = 150. The amplitudes of MLK and FEX
at their zero crossing points of 341 nm and 213.6 nm,
respectively, were used for quantitation, as shown in Fig. 4.
Third derivative method This technique involves theconversion of a normal zero-order absorption spectrum
to a 3D spectrum [30]. The zero-crossing point in thederivative spectrum corresponds to the λmax of a compound, permitting direct estimation without interference.
Here, 3D spectra were obtained using Δλ = 16 nm andSF = 6000. The peak amplitudes at 295, 336.4, and 346 nm
for MLK and 235 nm for FEX showed good linearity andwere used for the assay, as shown in Fig. 5.
Ratio difference method In this method, the ratio spectra were obtained by dividing the standard spectra of one
analyte by a fixed concentration of the other analyte asa divisor [30]. The difference in amplitudes at two wavelengths was proportional to the analyte concentration but
was zero for the divisor. For MLK, the difference between
250 and 245 nm, and for FEX, the difference between 227
and 217 nm showed good linearity and sensitivity andwere used for the assay, as shown in Fig. S1.
Method validation

The proposed micellar UPLC and spectroscopic methods
were validated as per the ICH recommendations [31].
System suitability testing

To evaluate system performance, system suitability
parameters such as retention period (­tR), number oftheoretical plates (N), and resolution ­(Rs) were investigated as system appropriateness criteria. The optimized

3.317 ± 0.028 4.281 ± 0.26

0.999

99.67

Intercept

Determination coef‑
ficient ­(r2)

Accuracy (%R)

1.37

LOQ (μg/mL)

0.264

0.079

1.16

0.452

1.43

98.94

0.999

1.53

LOD (μg/mL)

3–50

341

MLK

3–50

295

MLK

3–60

235

3–50

ΔP 250–245

MLK

0.0482 ± 0.0002 0.0391 ± 0.0003 0.0509 ± 0.0004

3–50

346

FEX

0.0346 ± 0.0003

3–60

ΔP 227–217

FEX

Ratio Difference (RD)

2.508

0.828

0.34

0.36

100.560.99962.037

0.6721

1.37

0.54

99.54

0.9998

1.781

0.588

0.85

0.62

101.280.9998

1.382

0.456

0.87

0.71

100.910.9999

1.414

0.467

0.91

0.68

100.270.9999

2.357

0.778

0.54

1.06

98.62

0.9998

2.458

0.811

0.83

0.64

99.37

0.9996

2.586

0.853

1.16

0.52

101.820.9997− 0.0041 ± 0.0001 0.1905 ± 0.0072 − 0.0362 ± 0.0091 0.0105 ± 0.0054 0.0698 ± 0.0068 0.0356 ± 0.0092 − 0.0544 ± 0.0125 0.0314 ± 0.0089

0.039 ± 0.0002

3–50

336.4

Third derivative (3D)0.0355 ± 0.0002 0.0513 ± 0.0003

3–60

213.6

FEX

Second derivative (2D)Spectroscopic methods

0.4582 ± 0.002 0.0109 ± 0.0027

Intermediate precision 0.23

Repeatability

Precision (%RSD):

9.672 ±0.11

Slope

1.2–312

1–260

230

230

FEX

Linearity range (μg/mL)

MLK

Micellar UPLC method

Wavelength (nm)Parameter

Table 1 The regression coefficients and validation outcomes about the quantification of MLK and FEX employing the proposed methodological approaches

katamesh et al. BMC Chemistry(2024) 18:149
Page 11 of 20

katamesh et al. BMC Chemistry(2024) 18:149

chromatographic conditions yielded an exceptional baseline resolution of the two analytes with negligible peaktailing. The chromatographic peaks exhibited high symmetry, ensuring accurate and precise quantification, as
indicated in Table S2.
Linearity range

UPLC calibration plots were meticulously establishedfor MLK concentrations ranging from 1–260 µg/mL and
FEX concentrations from 1.2–312 µg/mL. For spectroscopic methods, MLK had a linearity range of 3–50 µg/
mL and FEX had 3–60 µg/mL. Table 1 summarizes calculated regression characteristics. These high correlation
values confirm that drug concentrations and analyticalresponses are linearly related.
Sensitivity

Following the guidelines set forth by the ICH [31], theminimum concentrations required for reliable detection(limits of detection, LOD) and quantification (limits ofquantification, LOQ) were calculated. The low LOD and
LOQ values indicate the adequate sensitivity of the proposed methods for precisely detecting and quantifying
MLK and FEX, as shown in Table 1.
Accuracy and precision

Nine distinct measurements were taken at three concentrations (5, 10, and 20 μg/mL) for MLK and 60, 120, and220 μg/mL for FEX via the micellar UPLC method, while
spectroscopic methods were used (10, 20, and 50 µg/mL) and 6, 24, and 48 μg/mL) to assess the accuracy of
the MLK and FEX methods, respectively, as shown in
Table 1. To evaluate the precision of the experiment, its
repeatability and intermediate precision were examined,as shown in Table 1.
Selectivity

By testing various laboratory-prepared mixtures containing MLK and FEX separately within the linear range, theselectivity of the techniques was determined, and promising findings were achieved. Acceptable results wereachieved and can be found in Table S3.
Robustness

The method’s robustness was assessed by investigating
the impact of slight modifications in the mobile phase’spH (± 0.1), flow rate (± 0.1), and concentration (± 0.1%).
No discernible impact was found.
Application to tablet preparation

Using the provided techniques, the drug content of Montair-FX® tablets was successfully estimated. The results
met expectations and showed excellent accuracy with

Page 12 of 20

respect to the amount claimed on the label, with no evidence of excipient interference. Additionally, the F test,
Student’s t-test, and one-way ANOVA were used with a
95% confidence level to compare the results with thosefrom the reported methodology [25] statistically, there
were no appreciable differences between the suggested
and reported methods, as shown in Tables 2 and 3.
Comparison of calibration curve construction methods

UPLC and three spectroscopic methods (2D, 3D, andRD) were used. Direct side-by-side comparisons of key
validation parameters shed light on relative performancetrade-offs to help choose methods.
Spectral interferences are eliminated by selective
detection at specific retention times with UPLC chromatographic separation. Thus, 230 nm was chosen to
maximize MLK and FEX absorbance and sensitivity without matrix interference. However, spectral overlap hinders direct UV estimation. By computationally removing
background signals, derivative methods solve this problem. Each derivative order has mathematical properties
specific to analytes and matrices. In 2D, MLK and FEX
peak amplitudes at 341 and 213.6 nm were calibrated.
In 3D, MLK peak amplitudes at 295, 336.4, and 346 nm
were proportional to concentration. For estimation, the235 nm FEX peak was used. Finally, the RD is divided by
the standard analyte spectrum to reduce interference.
The defined absorbance ratios varied with concentration.
MLK used 250 nm to 245 nm amplitude difference, andFEX used 227 nm to 217 nm.
UPLC had the widest linear concentration range,
allowing for maximum calibration flexibility. Due todetector response constraints at higher concentrations,
all spectrophotometric methods had narrower ranges.
UPLC showed that FEX (1.2–312 μg/mL) had a wider linear range than MLK (1–260 μg/mL), suggesting strongerhydrophilic and H-bonding interactions with the stationary phase. Due to its stronger retention, FEX elutes
later than MLK under chromatographic conditions. FEX
can be quantified over a wider linear range before peakbroadening or distortion due to its higher retention andcolumn capacity factor. In contrast, MLK has a lower
column capacity and weaker retention. To prevent peakbroadening, which could compromise quantitation accuracy, MLK’s linear range was narrowed to lower concentrations. Spectroscopic techniques yielded narrower
linearity ranges of 3–50 µg/mL for MLK and 3–60 µg/mL
for FEX. As simpler instrumentation has slightly higher
detection limits, the linear ranges reach higher lower limits. The upper ends stay within Beer’s law for the cuvettepath length. The derivatization and ratio difference processes in the spectroscopic methods reduce backgroundnoise and improve analytical sensitivity, bringing MLK’s

1.73

F value (6.39)

1.76

1.66

0.48

variance

Student’s t test
(2.306)

1.36

99.74

1.29

Mean %

SD

1.2–312

2.58

0.41

100.04

5

1–260

5

FEX

Linearity range (μg/mL)

MLK

Micellar UPLC
methodnParameters

1.35

0.57

1.08

1.04

99.77

5

3–50

MLK

1.76

0.28

1.10

1.05

100.15

5

3–60

FEX

Second derivative

2.33

0.64

0.62

0.79

100.595

3–50 (295 nm)

MLK

Third derivative

Spectroscopic methods

3–50 (336.4 nm)

1.76

0.25

0.82

0.91

100.015

3–50 (346 nm)

1.03

0.30

1.51

1.23

100.41

5

1.76

2.27

0.36

0.60

99.31

5

3–60 (235 nm)

FEX

2.38

0.65

0.62

0.79

99.76

5

3–50

MLK

1.11

0.40

0.70

0.84

100.52

5

3–60

FEX

Ratio difference

Table 2 Comparative statistical evaluation of results obtained by the proposed and the reported methods for the analysis of MLK and FEX

–

–

1.47

1.21

100.185

12.5–37.5

MLK

–

–

0.627

0.79

100.32

5

150–450

FEX

Reported method[25]

katamesh et al. BMC Chemistry(2024) 18:149
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katamesh et al. BMC Chemistry(2024) 18:149

Page 14 of 20

Table 3 One-way ANOVA for comparative evaluation of proposed and literature-reported methodologies in pharmaceuticalformulation analysis with a 95% confidence interval
Component
MLK

FEX

a

Sum of squares

df

Mean squareF

P value

Between Groups

5.275

4.000

1.319

0.838 (2.866)a

0.517

Within Groups

31.46620.000

1.573

–

–
–

Total

36.74124.000––Between Groups

9.341

4.000

2.335

2.100 (2.866)a

0.119

Within Groups

22.24020.000

1.112

–

–

Total

31.58024.000–––Figures in parentheses represent the corresponding critical value of F at P < 0.05

linear range closer to FEX. In contrast, UPLC uses only
intact drug molecules’ native UV absorbance. Thus, theinherent sensitivity difference remains. This method
boosts MLK sensitivity.
In terms of sensitivity, UPLC found 3–fourfold lower
LODs for FEX, reaching 0.0079 µg/mL, almost 10 times
the initial targeted concentration. Optimized chromatographic separation methods can quantify ultra-trace
amounts. Moreover, spectrophotometric methods produced comparable LODs (0.5–0.8 μg/mL) suitable forpharmaceutical analysis. UPLC’s baseline resolution,
peak symmetry, and efficient separation in a packed column with small particles increase detection sensitivity.
This quantifies lower drug concentrations than cuvette
spectroscopy. The UV extinction coefficients of MLK and
FEX molecular structures affect UPLC detector response
factors. FEX outperforms MLK at 230 nm in UV absorption and detector signals at equal concentrations. This
explains why FEX had a fivefold lower LOD (0.0079 μg/mL) than MLK (0.452 μg/mL) using the same UPLC
instrumentation.
All methods had consistent accuracies and precisions
between 98 and 102% and RSD values within 1.53%.
This proves the proposed method’s quantitative performance meets the acceptance criteria. These methods
validate core data but have strengths and weaknesses.
UPLC separation resolves structurally similar components with unmatched selectivity. It also detects individual analytes in complex matrices using multi-wavelength
analyses. UPLC requires specialized infrastructure andvast method development expertise. Compared to othermethods, spectrophotometry is simple and cost-effective,
utilizing UV–visible instruments commonly found inlabs. However, spectral signal decomposition requires
complex mathematical calculations due to separation
capacity limitations.
Overall, while all the presented calibration methods
show fit-for-purpose validation characteristics, researchers must consider infrastructure constraints and specificquantification needs when selecting optimal techniques.

UPLC offers superior flexibility and sensitivity but
requires liquid chromatographic systems. Moreover,derivative and ratio spectrophotometry provides affordable, accessible alternatives, albeit with narrower ranges.
Thus, balancing key trade-offs can guide sustainable analytical choices.
Comprehensive greenness, blueness, and whitenessevaluation

Analytical methods’ sustainability credentials must be
evaluated since they offer priceless information about
their possible environmental and economic effects. But
the idea of sustainability is complex and includes manydifferent aspects, such as cost-effectiveness, safety concerns, waste reduction techniques, and environmental
responsibility (greenness) [8]. No single tool or assessment method can evaluate sustainability across all relevant parameters [30]. This study uses a synergistic
multi-tool approach to address this challenge by integrating complementary assessment strategies from different
perspectives. This strategic combination of evaluation
methods allows a more holistic and robust assessment of
the analytical techniques’ sustainability.
Evaluation of the method’s greenness profile

NEMI criteria The NEMI analysis offered an initial
screening of greenness deficiencies through a visual format separated into four quadrants [33]. One PBT (persistent, bioaccumulative, and toxic), two hazardous, threecorrosive, and four waste products are the contents ofthese four quadrants. The green quadrant signifies thefollowing: (1) the reagents used are not PBT-classified
according to the Toxic Release Inventory (EPA-TRI) of
the Environment Protection Agency; (2) the employed
substances are deemed nonhazardous and are thus notincluded in the TRI list; and (3) the pH of the medium
is between 2 and 12. Additionally, waste generation isless than 50 g (Fig. S5). For the suggested methodology,
NEMI pictograms were generated (Tables 4 and 5). A preliminary examination of the pictograms revealed that thekatamesh et al. BMC Chemistry(2024) 18:149

Page 15 of 20

Table 4 Comparative study of the proposed and reported UPLC methods
Parameter

Proposed method

Reported method
[25]

88

75

NEMI tool

ESA toolComplex GAPI tool

AGREE tool

BAGI tool`
RGB 12 algorithm

recommended method satisfied all the NEMI criteria, as
evidenced by the four green quadrants.
ComplexGAPI criteria NEMI allows introductorygreenness examination, but Complex GAPI allows a morecomprehensive semi-quantitative evaluation [34]. It isbetter than the original GAPI measure because it includes
a hexagon-shaped area showing pre-analysis phases andstages, as illustrated in Fig. S6. This advanced tool handles all steps of the analytical process, from collecting andtransporting samples to securing, storing, preparing, andanalyzing them [34]. Complex GAPI offers easy-to-use
pictogram software. Green icons and a low E-factor value

show that this study’s methodologies are very green. Withan impressive E factor of one, these methods generate
minimal waste, improving sustainability and the environment. This remarkable accomplishment underscores thesuperiority of the developed approaches over the reported
techniques in terms of greenness, as summarized inTables 4 and 5. Despite its benefits, ComplexGAPI’s scopeis limited to environmental criteria, leaving waste prevention, energy efficiency, and renewable resource incorporation unaddressed. Overreliance on ComplexGAPI alone
may result in an incomplete and narrow assessment.
Complementing ComplexGAPI analysis with quantitative
assessment methods can overcome this limitation andkatamesh et al. BMC Chemistry(2024) 18:149

Page 16 of 20

Table 5 Comparative study of the proposed and reported spectrophotometric methods
Parameter

NEMI tool

ESA tool ComplexGAPI tool

Proposed method

92

Reported method [15]84

Reported method [16]85Reported method [17]79Reported method [18]82

Reported method [19]82

Reported method [20]83AGREE tool

BAGI tool

RGB 12 algorithm

katamesh et al. BMC Chemistry(2024) 18:149

provide a more complete evaluation. ComplexGAPI andother complementary tools will bridge research gaps andaddress the constraints of using a single assessment technique in this integrated approach.
AGREE criteria The AGREE metric, which takes into
account all 12 GAC philosophies, is a beneficial quantitative method for measuring greenness [35]. This enables thorough assessment grounded in widely accepted
GAC criteria. A key advantage of AGREE is its flexibility
through customizable weighting of these diverse parameters. The user-friendly software converts the 12 inputs
into a single score from 0 to 1, visualized on a coloredpictogram for rapid interpretation. Dark green indicatesexcellent greenness, while dark red signifies major deficiencies. For this study, the proposed methodologies
exhibited exemplary greenness, attaining impressivescores of 0.86 and 0.9 for the UPLC and spectrophotometric techniques, respectively underscores the superiority ofthe developed approaches over the reported techniques,as shown in Figs. S7–S15. These scores signify their efficient alignment with green principles. Visualizations inTables 4 and 5 show how environmentally friendly the
proposed methods are, proving that they follow sustainable analytical practices. Since the AGREE tool only evaluates environmental criteria related to green chemistry
principles, it ignores safety, analytical performance, andcost-effectiveness assessments. Thus, AGREE alone may
provide an incomplete and narrow evaluation. AGREE
should be used with complementary tools that evaluate
these additional sustainability aspects to overcome theselimitations and ensure a complete assessment.
ESA criteria The ESA is a widely employed green metric for assessing the environmental impact of analyticalprocedures. It offers several advantages over other assessment tools, primarily due to its user-friendly nature andability to quantitatively evaluate diverse aspects of environmental sustainability [36]. The ESA score is derived bydeducting penalty points from a maximum of 100, basedon various factors inherent to the analytical workflow,such as reagent quantities, hazardous properties, energy
consumption, and waste generation. Consequently, higherscores, approaching the maximum of 100, are indicativeof more environmentally responsible and greener methodologies [37]. In our study, we have calculated the ESA
values for the proposed analytical approaches and compared them with those of previously reported methods, aspresented in Tables 4 and 5. The results demonstrate thatour developed methods exhibit a superior greenness profile, with the highest ESA score of 88 and 92 for UPLC and
Spectrophotometric methods respectively. This remarkable score classifies our proposed techniques as excellent

Page 17 of 20

green analytical methods, surpassing the environmental sustainability credentials of existing methodologies.
While the ESA tool is a valuable and widely used metricfor assessing the greenness of analytical methodologies,
it is important to recognize its limitations and potential
disadvantages including oversimplification, limited scope,subjectivity in scoring, lack of weighting, and static set ofcriteria and scoring rules, which may not evolve or adapt
to emerging sustainability concerns, new technologies, orchanging regulatory frameworks. To address these limitations, it is often recommended to complement the ESA
tool with other complementary assessment methods.
BAGI criteria

Unlike predominantly greenness-focused tools, therecently introduced BAGI metric delivers a quantitative
estimation of an analytical method’s “blueness”, defined as
its real-world fitness for purpose based on critical practical criteria [38]. BAGI facilitates an inclusive evaluationof an analytical process’s suitability or "blueness" by considering ten crucial factors: the kind of analysis, the number of analytes used, the instrumentation employed, thesample productivity, the prerequisites for sample preparation, the number of samples examined in an hour, thereagents and materials demanded, the need for pre-concentration steps, the automation level, and the quantityof sample needed. Each of these ten factors is assigned ascore ranging from 1 (lowest) to 10 (highest). All ten criteria contribute to the final BAGI score, which is determined by taking the geometric mean of those scores.
More relevance, functionality, and fit for the intended
purpose are characteristics of an analytical method witha higher BAGI score. In this study, UPLC and spectrophotometric protocols obtained high BAGI scores of
82.5 and 90, respectively, indicating excellent real-world
applicability, high throughput, automation potential, andlow operation costs. The short analysis times of 1.67 min(FEX) and 3.54 min (MLK) in UPLC and straightforward
measurements in derivative spectrophotometry offer
significant practical advantages. Moreover, the use of an
eco-friendly aqueous micellar mobile phase minimizes
material requirements and waste generation, echoing
GAC principles within the applied analysis. Hence, the
BAGI assessment confirmed the outstanding blueness ofthe developed methods. However, since BAGI specificallyfocuses on practical criteria, it does not provide holistic
sustainability quantification. Therefore, we additionallyemployed the RGB12 algorithm to evaluate composite
analytical sustainability considering greenness, performance, and practicality. The consistently high scores
across tools verify this method’s merit as an implementable green chemistry alternative.

katamesh et al. BMC Chemistry(2024) 18:149

Page 18 of 20

RGB12 criteria

Significance of the multi‑tool assessment strategy

In the evaluation of the overall sustainability of the proposed methods, termed "whiteness," the RGB12 approach
was employed as an effective algorithm for quantitatively
assessing the sustainability level of analytical techniques[8]. It is a quantitative assessment metric that is easy touse for evaluating whiteness. This tool determines thedegree of sustainability concerning whiteness assessment
and assesses methods based on the 12 WAC considerations [39]. Twelve separate algorithms, categorized as"red," "green," and "blue," make up the RGB12 algorithm,
where each group consists of four algorithms. The green
subgroup (G1-G4) includes essential GAC parameterssuch as toxicity, amount of waste and reagent, energy
conservation, influence on humans and animals, andgenetic alterations. The red subgroup (R1-R4) focuseson validation parameters such as application scope,
accuracy, LOD, precision, and LOQ. The blue subgroup(B1-B4) concerns practical and economic necessities,
cost-effectiveness, and time efficiency. The RGB12 algorithm exemplifies adherence to WAC principles by summing the scores of the method in each of the three-color
domains to obtain the ultimate "whiteness" value. As
illustrated in Tables 4 and 5, the proposed methods attainremarkable whiteness values of 88.1 and 89.8 for the
UPLC and spectrophotometric methods, respectively, incontrast to the lower values observed for the reported
methods. Comprehensive RGB12 algorithm evaluation
confirms the proposed methodologies’ exceptional performance, demonstrating their sustainability and superiority over existing methods, as illustrated in Fig. S16 andFig. S17. This assessment further highlights the proposed
approaches’ remarkable performance, dependability, andeconomic viability, rendering them attractive options fordiverse analytical applications. The RGB12 tool’s strengthis its ability to evaluate an analytical method’s alignment
with sustainable analytical chemistry principles holistically and multidimensionally, surpassing other assessment methods. RGB12 assesses all relevant sustainability
parameters, unlike NEMI, ComplexGAPI, AGREE, ESA,
and BAGI, which focus on specific aspects. RGB12 complements these other tools by quantifying how well ananalytical approach follows sustainable analytical practices. By using RGB12 and other assessment metrics, thisstudy was able to thoroughly assess the sustainability ofthe proposed analytical methods. This multipronged
approach mitigated the limitations of any single assessment technique, ensuring a more balanced and unbiased
evaluation. This research’s systems-oriented strategy,
which integrated multiple complementary assessment
tools, shows how to evaluate analytical method sustainability thoroughly and rigorously without biases or blind
spots.

The study demonstrated the benefits of using multiple
complementary tools (NEMI, ComplexGAPI, AGREE,
ESA, BAGI, RGB12) to evaluate the sustainability of analytical methods across various dimensions. These tools
holistically assessed greenness (environmental impact),
blueness (practicality/cost-effectiveness), whiteness(overall sustainability including performance and safety),and other metrics. The developed analytical procedures
showed superior sustainability compared to existing
methods based on factors like reduced toxicity, waste
minimization, material efficiency, high throughput, automation potential, low cost, and excellent analytical performance. The combined qualitative and quantitativeevaluation confirmed the new methods as highly sustainable and implementable best practices that align wellwith the principles of green analytical chemistry acrossthe environmental, economic, and performance domains.

Conclusion
Driven by sustainable development goals, we successfully
developed and validated an eco-friendly micellar UPLC
method and complementary spectrophotometric techniques for the pharmaceuticals MLK and FEX. Comprehensive validation demonstrated excellent performancefor simultaneous quantification under optimized conditions, with high linearity, accuracy, precision, sensitivity,and selectivity. The successful application of these methods to pharmaceutical samples further proved the suitability of the methods for routine analysis. Additionally,
extensive greenness, blueness, and whiteness assessments
using tools such as NEMI, ComplexGAPI, AGREE, ESA,BAGI, and RGB12 verified the outstanding sustainability
metrics, efficacy, and practical functionality. The use ofan eco-friendly aqueous micellar mobile phase reduced
solvent usage and waste generation by more than 50%
compared to the use of existing methods, echoing green
analytical principles. Short run times of less than 3.5 min
and operational simplicity offered significant practicaladvantages, as reflected by high BAGI scores of up to
82.5 and 90 for UPLC and spectrophotometric methods, respectively. Furthermore, the RGB12 sustainability
indices up to 88.1 and 89.8 for the UPLC and spectrophotometric methods, respectively, confirmed the excellent composite performance across critical parameters.
Therefore, the developed methods align analytical science with sustainable practices by reliably integrating
environmental and economic aspirations. This paper
reports the successful demonstration of implementablegreen analytical techniques balancing efficacy and ecofriendliness pioneer pathways for widespread adoption
in pharmaceutical quality control. We envision that thiswork will motivate analytical scientists to proactively

katamesh et al. BMC Chemistry(2024) 18:149

Page 19 of 20

embed holistic sustainability considerations when developing new solutions.

5.

Supplementary Information

6.

The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​024-​01254-8.
Supplementary Material 1.
Author contributions
N.S.k. Supervision, methodology, and visualization. A.E.F.A Design of the work,
investigation, writing-original draft, and writing review and editing. M.K.H
Analysis of data and writing review and editing. MAA. Interpretation of data
and figures preparation S.A.M: Supervised analysis procedures and carriedout sample preparation. All the authors read, reviewed, and approved themanuscript.
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB). This work was not funded by a third party.
Availability of data and materials
The corresponding author will provide the datasets created and/or analyzed
during the current study upon reasonable request.

7.

8.

9.

10.

11.

Declarations
Ethics approval and consent to participate:Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy (Girls),
Al-Azhar University, Cairo 11754, Egypt. 2 Analytical Chemistry Department,
Faculty of Pharmacy, October 6 University, 6 October City, Giza 12585, Egypt.
3
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy,
Al-Azhar University, Assiut Branch, Assiut 71524, Egypt.
Received: 12 March 2024 Accepted: 23 July 2024

12.

13.
14.

15.

16.
17.

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