(2024) 18:143
El‑Masry et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01242-y

BMC Chemistry

RESEARCH

Open AccessA dual methodology employing
ion‑pair chromatography and built‑in UV
spectrophotometry for quantifying recently
approved combination of mometasone
and indacaterol in a novel combined metereddose inhaler: assessing the greenness, carbon
footprint, blueness, and whitenessAmal A. El‑Masry1* , Ahmed Emad F. Abbas2 and Yomna A. Salem3*   

Abstract
Developing analytical techniques that align with green and sustainable chemistry principles is crucial in today’s
scientific landscape. This work introduces two innovative approaches for the simultaneous quantification of inda‑
caterol (IND) and mometasone (MOM), a recently approved combination therapy for chronic obstructive pulmonary
disease. These methods—rapid isocratic ion pair chromatography (IPC) and UV–visible spectrophotometry—demon‑
strate improved environmental sustainability, cost-effectiveness, and versatility compared to existing techniques. The
optimized 4-min IPC method achieved excellent resolution (retention times 2.18 ± 0.1 min for IND and 3.95 ± 0.1 min
for MOM), peak symmetry, and sensitivity. It utilizes a low-cost ion pair mobile phase of acetonitrile and acidified watercontaining 0.025% sodium dodecyl sulfate (50:50% v/v), making it suitable for laboratories with standard chroma‑
tographic instruments. The spectrophotometric approach offers two procedures: first derivative and ratio derivative
methods. These serve as simplified, low-cost alternatives for resource-limited laboratories without access to advanced
instruments. Both techniques feature simplified protocols that minimize extraction and fractionation steps. Compre‑
hensive validation confirmed outstanding accuracy (98–102%) and precision (%2 <). Sustainability assessments using
ComplexGAPI, AGREE, carbon footprint, BAGI, and RGB12 tools demonstrated enhanced environmental performancecompared to existing methods. The IPC and spectrophotometry methods achieved greenness scores of 0.81 and 0.85,
respectively, surpassing the 0.63–0.67 range of reported techniques. Additionally, they showed lower carbon foot‑prints of 0.035 and 0.022 kg CO2 equivalent emissions per sample, compared to 0.079–0.092 kg for conventional
procedures. The application of novel "blueness" and "whiteness" concepts using BAGI and RGB12 algorithms further*Correspondence:Amal A. El‑Masry
dr.Amal90@mans.edu.eg
Yomna A. Salem
yomnasalem26@gmail.com
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://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

El‑Masry et al. BMC Chemistry(2024) 18:143

Page 2 of 15

confirmed superior sustainability, with scores of 87.5 & 90 for blueness and 88.1 & 89.8 for whiteness. Successfully
applied to quantify IND and MOM in combined capsules, this work provides a model for eco-friendly pharmaceuticalanalysis that maintains high analytical reliability while improving sustainability metrics.
Keywords Mometasone, Indacaterol, Ion-pair chromatography, Eco-friendly spectroscopy, Greenness bluenesswhiteness assessment

Introduction
In recent years, researchers have faced the complex challenge of balancing efficacy, eco-friendliness, and practicality when developing analytical methods [1–3]. As aresult, principles of white analytical chemistry (WAC)
and green analytical chemistry (GAC) have gained significant attention as a means to achieve this equilibrium andcontribute to a more sustainable future in quality control practices [4–6]. One such combination that calls forgreener and more sustainable quality control practices isthat of a novel anti-chronic obstructive pulmonary disease (anti-COPD) combination of indacaterol (IND) andmometasone (MOM).
COPD represents a significant global health challenge,
ranking as the third leading cause of death worldwide
after heart disease and stroke [7]. Over recent decades,
COPD treatment has evolved substantially with theintroduction of potent inhaled corticosteroids (ICS)
and long-acting β2-adrenoceptor agonists (LABAs)[8]. Combination therapy using ICS such as MOM and
LABAs like IND in a single inhaler has become a mainstay of COPD management, offering superior efficacy
and improved adherence compared to ICS monotherapy[9]. Despite these therapeutic advancements, analyticalmethods for quantifying these drugs have lagged behind.
As of 2023, only three published methods were available, each with notable limitations especially concerning long separation time, lower sensitivity, and use ofhigh amounts of hazardous organic solvents that hinder
their use in routine quality control. The first method [10]utilizes micellar liquid chromatography, which suffersfrom issues with column stability over prolonged use andrequires careful control of mobile phase conditions likepH in addition to the requirements for specialized instrumentation, and expensive reagents that may not be accessible to all laboratories. The second method [11] uses acomplex gradient elution program and 45-min column
equilibration before analysis thus not amenable to highthroughput analysis. Finally, the third method [12] also
employs a complex gradient elution program in addition
to an expensive ultra-performance liquid chromatography that is inaccessible to most laboratories. However,
two additional methods were published in 2024 [13, 14],they still rely on non-green solvents and complex instrumentation, rendering them unsuitable for routine quality
control practices. Thus, there is a need for a more green,
simple, rapid, and cost-effective quantification technique
for these critical inhaled drugs.
Our present work aims to develop two new analyticalmethods to quantify IND and MOM in their combined
metered dose inhaler formulation that overcomes theselimitations through a dual methodological approach
employing ion pair chromatography (IPC) for equippedlaboratories and UV–visible spectrophotometry forunder-resourced laboratories. IPC is a well-establishedmode of high-performance liquid chromatography thatallows excellent separation through the formation of ion
pairs between ionizable analytes and oppositely charged
ion-pairing reagents, improving compound separationand peak resolution with consistently reproducible performance and better peak shape [15–17]. Our optimized
IPC method using widely available sodium dodecyl sulfate (SDS) as an ion-pair reagent enables rapid quantification in only 4 min. The short analysis time confers theadvantages of high throughput, low organic solvent use,and excellent column stability for greenness and costeffectiveness. UV-spectrophotometry serves as an excellent alternative technique given its low operational costs
and suitability for laboratories with limited resources,
supporting sustainability goals. By requiring only inexpensive reagents and basic equipment like cuvettes andlight sources, with minimal hazardous waste generation,it aligns well with the principles of both GAC and WAC[18, 19]. Our optimized UV method uses solvent of water(80%) and ethanol (20%) as the eco-friendly solvent,offering a practical, economical, and sustainable quantification technique.
The core objectives of this study are six-fold: First, todevelop an eco-friendly isocratic chromatographic analytical method for the simultaneous quantification of IND
and MOM using the IPC method that offers short analysis times, high selectivity, excellent resolution, peak symmetry, and optimal linearity and sensitivity. Second, todevelop a complementary green spectroscopic approachfor quantifying IND and MOM to provide an easy-touse and affordable option in laboratories lacking accessEl‑Masry et al. BMC Chemistry(2024) 18:143

to expensive chromatographic instrumentation. Third,
to comprehensively assess the greenness of the proposed
methods using established tools like AGREE (Analytical Greenness Metric), Complex GAPI (Complementary
Green Analytical Procedure Index), and carbon footprint. This will verify enhanced environmental benefits
compared to existing methods. Fourth, to introduce andapply the novel concepts of "blueness" and "whiteness"
assessment using the recently introduced Blue Applicability Grade Index (BAGI) and Red–Green–Blue 12 (RGB12) algorithms. This will demonstrate the superiority ofthe proposed approaches over reported methods in termsof greenness, analytical performance, cost-effectiveness,and practical utility. Fifth, to demonstrate the applicability of the developed protocols by analyzing IND and
MOM in their commercially available combined dosage
form. Finally, to present an exemplary model for environmentally friendly pharmaceutical analysis that does notcompromise on accuracy or performance establishing astandard for sustainable quality control practices.

Experimental
Reagents and materials

The solvents employed were of HPLC grade, and all thecompounds were of analytical reagent grade. Sodium
dodecyl sulphate (SDS, 98.5%), triethylamine, trimethylamine, acetonitrile, ethanol, methanol, and orthophosphoric acid (OPA) were acquired from Sigma-Aldrich(Steinheim, Germany). Ultra-pure water was obtained
from a Milli-Q water purification system (Millipore,
USA) and used throughout the analysis.
Samples
Reference standards• Reference standard of MOM with lot no. M-0190223
and a purity of 99.59% was kindly gifted by Global
Napi Pharmaceuticals, 6-October City, Cairo, Egypt.
• Reference standard of IND with lot no. I0009301
and a purity of 99.65%, was generously provided by
Egypt’s National Organization for Drug Control andResearch.
Pharmaceutical sample

Thepharmaceuticalformulation,
ATECTURE­breezhaler® Novartis Pharma Stein AG (Switzerland),batch number BCND3 was purchased from a local pharmacy. As per the label claim, each capsule was formulated to contain 150 µg IND and 160 µg MOM.

Page 3 of 15

Apparatus• For the liquid chromatography, separations were performed on a Perkin Elmer Series 200 system (PerkinElmer Inc., Massachusetts, USA) coupled with a
Rheodyne model 7725i injector (20 μL sample loop)
and a UV/VIS detector set to 259 nm. Data acquisition and processing were done using TotalChrom
Workstation software. A 0.45 μm membrane filter(MilliporeSigma, Cork, Ireland) was employed for filtration of the mobile phases prior to use. Degassing
was carried out in an ultrasonic bath (model SS 101
H 230, Branson Ultrasonics Corporation, USA). pH
measurements were done using a P-901 pH meter(A&E Laboratory, UK).
• The spectrophotometric analysis was conductedusing a Shimadzu UV-1601 PC double-beam UV–
visible spectrophotometer (Shimadzu Corporation,
Kyoto, Japan) equipped with a pair of matched 1 cm
quartz cells. The spectrophotometer was operatedusing in-built UVProbe software version 2.21 witha scanning speed of 2800 nm m
­ in−1 (fast scan) andbandwidth of 2 nm.
Standard solutions

Stock solutions were prepared separately for the spectrophotometric and IPC methods. 15 mg of IND and 16 mg
of MOM reference standards were accurately weighedand quantitatively transferred into a 100 mL volumetricflask. The contents of the flask were dissolved in a solvent of water (80%) and ethanol (20%), sonicated for 5min, and then diluted to volume with the same solvent
to obtain stock solutions of 150 μg/mL for IND and 160
μg/mL for MOM. The stock solutions were then appropriately diluted using the same solvent (spectrophotometry) or mobile phase (IPC) as diluents to obtainworking standard solutions covering the desired calibration ranges. All stock solutions were stored at 4 °C inamber-colored containers to protect them from light andwere found to be stable during the study period.
Procedures
IPC method chromatographic conditions

A Shim-pack cyano column (150 mm × 4.6 mm; 5 μm
particle size) was used for the separation of IND and
MOM at ambient temperature (25 °C). The optimized
mobile phase consisted of 50% v/v acetonitrile and 50%
v/v acidified deionized water containing 0.025% w/v SDS.
The pH of the aqueous component was adjusted to 3.0

El‑Masry et al. BMC Chemistry(2024) 18:143

using a dilute OPA solution prior to mixing. The resulting mobile phase was filtered through a 0.22 μm membrane filter and ultrasonically degassed before use. A flowrate of 1 mL ­min−1 was maintained during the analysis.
Analyte detection was carried out at 259 nm. Under
these chromatographic conditions, efficient separation of
MOM and IND was achieved within 4 min. The mobile
phase was found to retain stability for two weeks duringstorage at room temperature.
Spectroscopic method

UV–Vis’s spectra of IND and MOM solutions wereacquired from 200–400 nm using a solvent of water (80%)
and ethanol (20%) as blank and the overlaid zero-orderabsorption spectra were imported into built-in spectrophotometer software (UVProbe) for further data analysis
without the need for complicated software.
Construction of calibration curves
IPC method

Appropriate volumes of standard drug solutions werediluted with mobile phase to yield concentrations in therange of 1–10 μg/mL for IND and MOM. The solutions
were subsequently examined by injection into the chromatographic system and eluting them using the mobile
phase while adhering to the aforementioned chromatographic parameters. The calibration charts were generated by the process of plotting the peak area against therelevant concentrations, followed by the calculation of
linear regression equations.
Spectroscopic method

A series of 10-mL volumetric flasks were prepared withaliquots of standard IND and MOM solutions ranging
from 1.1–32 μg/mL for Mom and 1–30 μg/mL for IND,
then filled to the required volume with a solvent of water(80%) and ethanol (20%). The absorption spectra of the
prepared drug solutions were measured over 200–400
nm using this solvent as a blank.
First derivative method (1D) The stored spectral data wasprocessed to obtain the 1D spectra using the built-in UVProbe software with wavelength difference (Δλ) = 20 nm
and scaling factor (SF) = 10. The peak amplitudes at
251.62 nm for IND and 275.67 nm for MOM were measured. Calibration curves were constructed by plotting thepeak amplitudes versus the corresponding concentrations
in μg/mL and the relevant regression equation was calculated.

Page 4 of 15

Ratio derivative method (RD) Using the built-in UVProbe software, the ratio spectra of MOM are acquiredby dividing the recorded MOM’s zero-order absorption
spectra by the IND divisor spectrum (1 μg/mL). Similarly, the ratio spectra of IND are acquired by dividing
the recorded IND’s zero-order absorption spectra by
the MOM divisor spectrum (1 μg/mL). The obtained
ratio spectra were processed to obtain the first derivative of the ratio spectra (1DD) with (Δλ) = 20 nm and(SF) = 100. The peak amplitudes at 310.49 nm for IND
and 280.64 nm for MOM, respectively, were measured.
Calibration curves were constructed by plotting thepeak amplitudes versus the corresponding concentrations in μg/mL and the relevant regression equation was
calculated.
Analysis of laboratory‑prepared mixtures• IPC method
Aliquots of IND and MOM keeping the pharmaceutical ratio of 1:1.06 were added to 10 mL volumetricflasks, which were then completely filled with mobile
phase to yield different concentration ratios of the two
drugs covering the linearity range. The solutions weremixed well and analyzed under optimized IPC conditions. The concentrations of IND and MOM were
determined from respective regression equations.
• Spectroscopic method
Aliquots of IND and MOM keeping the pharmaceutical ratio were added to 10 mL volumetric flasks, whichwere then completely filled with a solvent of water(80%) and ethanol (20%) to yield different concentration ratios of the two drugs covering the linearity range.
The absorption spectra of these laboratory mixtures
were recorded as described earlier. Concentrations of
IND and MOM were determined using the respective
spectroscopic methods.
Analysis of the studied drugs in their co‑formulated inhaler
capsuleTwenty Atectura B­ reezhaler® inhaler capsules (labeled
to contain 150 μg of IND and 160 μg of MOM per capsule) were emptied and mixed thoroughly. A precisely
measured amount of powder, equal to the weight often capsules, was placed into a volumetric flask with acapacity of 100 mL. Approximately 50 mL of a solventof water (80%) and ethanol (20%) was added and sonicated for 15 min to extract the drugs into the solution.

El‑Masry et al. BMC Chemistry(2024) 18:143

Page 5 of 15

Fig. 1 Chemical structure of a MOM and b IND

The content was mixed well, passed through a membrane filter with a pore size of 0.45 μm, and appropriately diluted with the same solvent to the mark. Suitablealiquots were transferred from the clear filtrate into 10
mL volumetric flasks and diluted with the same solvent(spectrophotometry) or mobile phase (IPC). The steps
were carried out in accordance with the previously outlined procedures to ascertain the concentration of thetwo medicines by employing the regression equations.

Results and discussion
In the current study, the approach was to produce amore effective simultaneous determination of antiCOPD medicines, IND, and MOM (Fig. 1) by employing IPC and complementary spectroscopic methods
with higher sensitivity, low cost, and minimum use ofhazard solvents that offer applicable methods for routine quality control analysis. Furthermore, a variety ofcomplementary sustainability assessment techniques
were used to test the presented methods’ green andwhite attributes. The outcomes demonstrated how
well the recommended approach performed according to the sustainable analytical chemistry parametervalues. The developed IPC method provides efficient
isocratic separation between IND and MOM within alower elution time of only 4 min in synthetic mixturesand co-formulated metered dose inhalers, as shown in
Fig. 2, which provides numerous sustainability advantages including 1—higher sample throughput thatmaximizes information output while minimizing overall run costs and energy consumption. 2—less solvent
usage that reduces chemical waste generation and disposal burdens. 3—enhanced durability as rapid quantifications extend column lifespan and periods between
maintenance, that resilience enables longer operational periods and replacement cycle times. 4—lower
energy necessary and less electricity and resources aredemanded for the same analytical workload. 5—substitution of organic solvents by alternative ion pairingFig. 2 IPC chromatogram demonstrating the separation of IND
and MOM. a IND (7.5 μg/mL, 2.18 min) and MOM (8.0 μg/mL,
3.95 min) in the synthetic mixture, b IND (1.5 μg/mL, 2.24 min) MOM(1.6 μg/mL, 4.03 min) in the inhaled capsule

El‑Masry et al. BMC Chemistry(2024) 18:143

compounds minimizes hazardous waste and toxicity
risks. In the proposed method the SDS was chosen as
the ideal ion pairing agent after evaluation of several
options. SDS offered unique advantages including
enhanced resolution and separation between IND and
MOM based on altered hydrophobicity and formation of neutral ion pairs with drugs functional groups,
improvement in peak shape and symmetry by controlling undesirable peak broadness and tailing, excellent
column efficiency and stabilization of column performance after prolonged use, low concentration sufficient for effect (0.025% v/v) which aligns method withgreen chemistry principles, environmentally-friendly
and less toxic compared to other conventional ion
pairing agents. Additionally, the complementary spectrophotometric techniques provide a greener, morecost-effective option for quantitation in resource-limited settings, with the sustainability metrics validating
their positive environmental profile.
IPC method development and optimization

All critical chromatographic parameters were systematically optimized to obtain adequate resolution, symmetric peak shapes, and short analysis time. The optimized
conditions are compiled in Table S1. The USP guidelines[20] include specific instructions for measuring chromatographic performance, including the number oftheoretical plates and the resolution. The results of thesemeasurements are provided in Table S1.
Selection of column

Different stationary phases were evaluated to find the
optimal column that provides good separation and peak
shape for IND and MOM. The columns tested included:
Zorbax SB C18 column (150 × 4.6 mm, 5 μm)—This
reverse phase column provided poor peak shape andco-elution of the analytes, Zorbax Eclipse XDB C8 column (150 × 4.6 mm, 5 μm)—The peak for IND was asymmetric using this column. The run time was also longer
compared to other columns over 12 min, Luna C18 column (150 × 4.6 mm, 5 μm)—Partial separation of IND
and MOM occurred but peaks were broad, and the total
run time was over 10 min. Shim-pack cyano column(150 mm × 4.6 mm, 5 μm)—This column provided goodpeak symmetry for both analytes and baseline separation
within only 4 min.
Selection of detection wavelength

Various wavelengths, including 200–300 nm, were tested.
Based on spectral evaluation and a better baseline,
259 nm was selected for detection allowing high sensitivity for both drugs.

Page 6 of 15

Optimization of mobile phase

The use of an ion pairing mobile phase offers numerous advantages over conventional organic solvents. They
serve to improve resolution between analytes with similar
physicochemical properties. The added ions in the agent
non-covalently interact with ionized sites on the analytes
and neutralize charges, altering the overall hydrophobicity and hydrophilic balance. This facilitates the separationof the ion pairs on common reverse-phase packing materials based on their new relative hydrophobicity. Various
compounds with amine or other ionizable groups can be
explored as possible ion-pairing agents for optimal selectivity and resolution [21–24].
Numerous mobile phase compositions were systematically assessed. Initial trials focused on three organic
solvents (methanol, ethanol, and acetonitrile) in combination with three different ion-pairing agents in acidified
water (triethylamine, trimethylamine, and SDS). These
components were mixed at organic solvent: aqueous
ratios of 40: 60, 45:55, 50:50, 55:45, and 60:40 v/v. Whenethanol and methanol were used, no separation occurred
for the IND and MOM. Moreover, when triethylamine
and trimethylamine were used gave a wide peak with along retention time. Ultimately acetonitrile and acidified
water containing SDS (50: 50% v/v) yielded satisfactory
resolution and a symmetrical peak with good clarity in
sufficient time (4 min), as shown in Table S1 and Fig. S1.
Effect of pH and buffer concentration

The effect of pH on the chromatographic behavior of IND
and MOM was studied using an increasing amount of
OPA between pH 2.5 to 6, as shown in Fig. S2. Additionally, the ionic strength of OPA (M) was studied between
0.05 and 0.3 M, as shown in Fig. S3. pH 3 and 0.1 M OPA
provided maximum theoretical plates and peak symmetry with well-resolved and sharp peak shapes as can beseen in Table S1.
Effect of concentration of SDS

Varying SDS concentrations between 0.015–0.03%
w/v were assessed. Lower SDS levels of around 0.015%
resulted in inadequate separation and peak broadening. Higher concentrations around 0.03% provided only
marginal improvements in resolution. Ultimately, 0.025%
SDS was determined to be the optimal concentration,
providing excellent peak shape and symmetry in a rapid
4-min isocratic run.
Effect of flow rate and column temperature

The flow rate varied from 0.8 to 1.2 mL/min, as shownin Fig. S4. Optimal separation with a significant reduction in retention time was achieved at a 1 mL ­min−1 flow

El‑Masry et al. BMC Chemistry(2024) 18:143

Page 7 of 15

and safety concerns associated with organic solvents,
while the small ethanol fraction ensures adequate solubility of the analytes. Furthermore, this solvent system iscompatible with standard laboratory equipment, making
it suitable for widespread adoption in various analyticalsettings. Its simplicity in preparation and stability during
analysis contribute to the method’s reproducibility androbustness. Overall, by selecting this water–ethanol mixture, we have developed a method that not only provides
optimal analytical performance but also supports sustainable practices in pharmaceutical analysis. This choicereflects our commitment to balancing scientific rigor withenvironmental responsibility in analytical chemistry.
Fig. 3 Zero order spectra of IND and MOM in a solvent of water(80%) and ethanol (20%) as blankrate. Similarly, the column oven temperature was varied
from 25° to 50 °C. While higher temperatures increased
theoretical plates, the tailing was also proportionately
increased. Hence, ambient temperature (25 °C) was
maintained giving the best peak shapes and resolution.
Spectrophotometric methods

The overlaid UV zero-order absorption spectra of IND
and MOM (Fig. 3) showed a lot of overlap, making theirdirect simultaneous estimation difficult. Hence, advanced
spectroscopic techniques were developed for their quantification. Two simple, economical, and accurate derivative and ratio derivative methods were developed.
Development and optimization

Selection of solvent The selection of an appropriate solvent is crucial for developing an effective and environmentally friendly spectrophotometric method. We conducted a comprehensive evaluation of various solvents tooptimize the solubility and absorption intensity of IND
and MOM. Our investigation encompassed pure solvents
including distilled water, ethanol, acetonitrile, and methanol, as well as aqueous buffers such as 0.1 M HCl, pH 8
borate buffer, 0.1 M NaOH, and pH 4 acetate buffer. Afterextensive testing, we determined that a mixture of 80%
water and 20% ethanol provided the optimal balance ofsolubility and absorption intensity for both drugs. Thissolvent combination demonstrated superior performancecompared to other tested options, yielding maximum solubility and the highest absorption intensity for IND and
MOM. The chosen solvent mixture aligns well with green
chemistry principles by minimizing the use of organic solvents while maintaining excellent analytical performance.
The high water content reduces environmental impact

Delta lambda and scaling factor for methods For derivative spectrophotometry, the derivative order, Δλ, and SF
were optimized to give sufficient sensitivity and selectivity at working concentrations. Δλ = 20 nm and SF = 10 For
the 1D method, and Δλ = 20 nm and SF = 100 for RD gavewell-defined peaks with the best signal-to-noise ratio.
Selection of divisors Different concentrations of MOM
and IND standards were evaluated as divisors to obtain
the optimized RD method. Concentrations of 1 μg/mL of
IND and 1 μg/mL of MOM gave the best recovery withthe highest sensitivity and reproducibility.
Method characteristics

First derivative method This technique helps remove
spectral interference by transforming the normal zeroorder absorption spectra into 1D signals. It takes advantage of the basic principle that derivative amplitudes of acomponent are directly proportional to its concentration,
while the influence of interfering drugs and excipients is
effectively abolished [25]. The critical aspect is that each1
D spectrum displays unique zero-crossing points where
the interference drug has no contribution. In the present
work, 1D spectra of IND and MOM were obtained byusing Δλ = 20 nm and SF = 10. For IND, the peak amplitude was selectively measured at its zero-crossing wavelength of 251.62 nm. Similarly, for MOM, quantification
was performed at 275.67 nm, as shown in Fig. 4. The
spectra were found to follow Beer’s law very well over thetherapeutic concentration ranges. Key attributes of thismethod are its rapid analysis speed, simple procedure,
sensitivity, cost-effectiveness, and avoidance of extensive separation steps—aligning excellently with GAC and
WAC principles.
Ratio derivative method In addition to the traditional
first derivative technique, the RD UV spectrophotometry
method was developed for resolving overlapping absorption spectra of the IND and MOM combination [26]. RD

El‑Masry et al. BMC Chemistry(2024) 18:143

Page 8 of 15

8.500

0.894
MOM (16 μg mL-1 )0.600

IND (15 μg mL-1 )

a

5.000

251.62 nm 275.67 nm
0.000

0.000

-0.600-5.000283.64 nm-1.146200.00-8.276200.00250.00300.00350.00400.00

Wavelength (nm)Fig. 4 First-order derivative spectra of IND and MOM where MOM
measured at 275.67 nm (at zero crossing for IND) and IND measured
at 251.62 nm (at zero crossing for MOM)

approach entails dividing the absorption spectrum of IND
or MOM (analyte) by a constant absorption spectrum of
MOM or IND (divisor), respectively. The constant divisor
spectrum used was 1 μg/mL for each drug. These vectorially divided analyte/divisor ratio spectra were then derivatized (1D) using a Δλ interval of 20 nm and a scaling factorof 100. In the resulting first derivative of the ratio spectra,
well-defined peaks were observed for IND at 286.49 nm
and MOM at 283.64 nm with zero crossing points ideal
for analytical measurement, as shown in Fig. 5. At thesewavelengths, only one particular drug component contributes such that simultaneous quantification of both isenabled without spectral interference. Calibration curves
were constructed by plotting peak amplitudes versus IND
or MOM concentrations. The regression equations exhibited excellent correlation coefficients (> 0.999), supporting the ratio derivative technique for selective and accurate quantification well within the therapeutic ranges. Key
benefits of the developed method include its simplicity,
precision, accuracy, and avoidance of tedious procedures
for removing background signals from excipients anddosing vehicles. The RD approach provided a rapid, economic, and green analytical strategy aligning with GAC
and WAC principles.
Method validation

The proposed IPC and spectroscopic methods were validated as per the ICH recommendations [27].
System suitability testing

To evaluate system performance, system suitability
parameters like the number of theoretical plates (N), Tailing factor (T), and resolution (­ Rs) were all investigated as

8.500

250.00b

300.00
Wavelength (nm)350.00400.00350.00400.00286.49 nm

5.000

0.000

-5.000-8.276200.00250.00300.00
Wavelength (nm)Fig. 5 a First derivative of the ratio spectra of MOM when 1 μg/mL
IND was used as the divisor and b first derivative of the ratio spectraof IND when 1 μg/mL MOM was used as the divisor

system appropriateness criteria. The optimized method
provided good baseline separation of the two drugs withminimal tailing, as indicated in Table S1.
Linearity range

Calibration curves were constructed covering suitable wide concentration ranges encompassing therapeutic doses of the IND and MOM combination. The IPC
method demonstrated excellent linear responses across
0.1–10 μg/mL for both IND and MOM. The regression statistics gave very high correlation coefficients(r > 0.999). Similarly, for the spectral techniques, the linearity range extended from 1–30 μg/mL for IND and
1.1–32 μg/mL for MOM. Again, the calibration curves
displayed regression coefficients greater than 0.999, supporting the strong linear relationships. The calculated
regression characteristics are summarized in Table 1.
The data were analyzed statistically giving small valuesof the standard deviation of residuals (Sy/x), intercepts(Sa), slopes (Sb), Percentage relative standard deviation

El‑Masry et al. BMC Chemistry(2024) 18:143

Page 9 of 15

Table 1 The Regression parameters and validation results for determining IND and MOM by the proposed methods
Parameter

IPC method

Spectroscopic methods
First derivative (1D)

MOM

IND

MOM

Ratio derivative (RD)
IND

MOM

IND

Wavelength (nm)

259

259

275.67251.62280.64

310.49Linearity range (µg ­mL−1)

0.1–100.1–10

1.1–321–30

1.1–32

1–30

Slope

62.34755.128− 0.007

0.01

0.065

0.083

Intercept

4.120

36.436− 0.016

0.013

0.024

− 0.017

correlation coefficients (r)0.99970.99980.99960.99980.99980.9999Accuracy (%R)

99.97

99.91

100.02

99.69

100.28100.11

 ­Repeatabilitya

0.531

0.832

0.366

0.774

0.682

0.755

Intermediate ­precisionb

0.732

0.965

0.348

0.675

0.985

0.857

LOD (μg/mL)

0.026

0.031

0.290

0.314

0.202

0.227

LOQ (μg/mL)

0.079

0.094

0.879

0.951

0.612

0.688

Sy/x

0.965

1.012

9.1 × ­10–4

1.2 × ­10–5

5.6 × ­10–4

8.12 × ­10–4

Sa

0.5

0.52

2.0 × ­10–5

5.00 × ­10–4

1.0 × ­10–5

0.01

0.099

0.103

–5

4.0 × ­10

–55.00 × ­10

2.2 × ­10–5

3.4 × ­10–5c% ­RSD

1.334

0.677

0.737

0.629

0.556

0.97

% ­Errord

0.471

0.239

0.301

0.257

0.227

0.395

Robustnesse (%RSD)

0.317

0.586

0.432

0.602

0.552

0.499

Precision (%RSD)

Sba

The intraday (n = 3), average of three responses of three different concentrations repeated three times on the same dayb

The interday (n = 3), average of three responses of different three concentrations repeated three times at three successive daysc

Percentage relative standard deviation(SD × 100/mean %R)√dPercentage relative error. = % RSD/ ne

Average of three determinations(%RDS), and Percentage relative error (% E), as shown in
Table 1.
Sensitivity

The limits of detection (LOD) and quantification (LOQ)
were determined using the slope and standard deviation of the intercepts of the regression lines as per ICH
recommendations.

LOQ =

Sa
× 10
b

LOD =

Sa
× 3.3
b

The low LOD and LOQ values indicate adequate sensitivity of the proposed methods to precisely detect andquantify IND and MOM, as shown in Table 1.
Accuracy and precision

To assess the method’s accuracy, nine distinct measurements were made at three various concentration levels. in
the IPC method, (1, 4, and 8 μg/mL) for each drug, while
spectroscopic methods used (5, 15, and 25 µg ­mL−1)for each drug. The excellent percentage recoveries (%R)
between 98 and 102% for repeated measurements displayed outstanding accuracy, as shown in Table 1.

Likewise for precision, standard deviations (SD) andpercentage relative standard deviations (%RSD) were
calculated based on replicate analyses at each concentration either on the same day (repeatability) or over threedays (intermediate precision). The very low SD (< 2%) and%RSD values (< 2%) highlighted the high degree of reproducibility and low variability around the mean, as shownin Table 1.
Selectivity

By testing various laboratory-prepared mixtures containing IND and MOM within the linearity range, the selectivity of the techniques was determined, and promisingfindings were achieved. Acceptable results were achieved,
as shown in Table S2.
Robustness

For the IPC method, the method’s robustness wasassessed by investigating the impact of slight modifications in pH (3.0 ± 0.2), proportion of acetonitrile (50 ± 2%v/v), and molar concentration of OPA (0.1 ± 0.02% w/v).
For spectroscopic methods, robustness was examinedby slightly altering experimental variables includingEl‑Masry et al. BMC Chemistry(2024) 18:143

Page 10 of 15

wavelength interval (0.9 nm instead of 1 nm), spectral
bandwidth (1.8 nm instead of 2 nm slit width), and Scan
speed (medium instead of fast scan). No discernible
impact was found in IPC and spectroscopic methods, as
shown in Table 1.

are not addressed. Thus, combining ComplexGAPI withadditional quantitative tools is recommended for a more
holistic assessment.

Application to tablet preparation

AGREE tool enables comprehensive evaluation acrossall 12 principles of GAC. Its flexibility, clear visual
outputs, and freely available software have promoted
widespread adoption [31, 32]. AGREE allows customized weighting of parameters and scoring from 0 to 1,
where 1 reflects full alignment with GAC principles.
The overall result is depicted on a clock-like graph,with the center color and score indicating greenness, as
shown in Fig. S5 to Fig. S10. Extensive AGREE assessments were performed for both the developed methods and the reported techniques. As shown in Table 2,
the proposed approaches demonstrated outstanding
greenness, achieving high scores of 0.7 (IPC) and 0.75
(Spectrophotometry) versus just 0.66, 0.63, and 0.64 forprior methods. The vivid green shading in the pictograms confirms the significant improvements in parameters like energy utilization, waste prevention, chemicalsafety, and use of renewable materials. This reinforces
the eco-friendly nature of the strategies. However,
AGREE predominantly focuses on environmental aspects of green chemistry. Critical sustainability
dimensions like economic feasibility, analytical performance, and robustness are not captured. Thus, pairing
AGREE with complementary tools is recommended fora more holistic evaluation, avoiding overemphasis onjust green metrics.

Using the provided techniques, the drug content of
ATECTURE ­breezhaler® capsules was successfully estimated. The results met expectations and showed excellent accuracy with the amount claimed on the label, withno evidence of excipients interfering. Additionally, using
t- and F-values with a 95% confidence level to compare
the results with those from the reported methodology[10] statistically, there were no appreciable differencesamong the suggested and reported methods, as shown in
Table S3.

Comprehensive greenness, blueness,
and whiteness evaluation
Assessing the sustainability of analytical methods
requires evaluating their environmental, economic, andperformance impacts across multiple criteria. No single
tool can comprehensively assess all relevant parameters[28, 29]. Therefore, this work employs a combined multitool approach for a more holistic evaluation from different complementary perspectives.
Evaluation of the methods’ green performance basedon ComplexGAPI criteria.

The innovative semi-quantitative ComplexGAPI tool
enables expanded assessment of green analytical procedure impacts beyond just the final analysis step. This
tool has gained significant recognition and acceptancewithin the chemical society due to its ability to enhancethe existing GAPI metric by incorporating a supplementary hexagonal field, based on the CHEM21 parameters.
This extended evaluation encompasses all stages of theanalytical method, such as sample collection, preservation, transportation, sample preparation, storage, andfinal analysis [30], as shown in Fig. S5. The user-friendly
shareware software generates a color-coded pictogramvisually summarizing the greenness at each stage ona scale from green to yellow to red. It also calculates acumulative E-factor indicating overall waste generation.
Our developed methods demonstrated excellent greenness as shown by the predominance of green icons anda very low E-factor of 1, reflecting the minimal environmental burden as summarized in Table 2. However,ComplexGAPI remains limited to primarily environmental criteria. Other key dimensions like waste prevention, energy efficiency, and renewable materials usage

Assessment of the methods’ green performance basedon AGREE criteria

Carbon footprint analysis

Carbon footprint analysis provides a quantitative evaluation of analytical methods’ environmental impacts
based on greenhouse gas emissions, reported as kilograms of C
­ O2 equivalent (kg ­CO2 eq) [33]. By accounting for electricity, transportation, materials usage, andwaste, it effectively complements other green chemistry tools lacking emissions quantification. Carbon footprints were calculated using the standardized equation[34]:

Carbon footprint(kg CO2 eq)
=Instrument Power(kW).Analysis time(h).
Emission factor(kg CO2/kWh)As shown in Table 2, the proposed IPC method
demonstrated a markedly lower carbon footprint ofCarbon footprint (kg­CO2 eq /sample)

AGREE tool

Complex GAPI tool

LOD (μg/mL)

0.035

Proposed IPC

IND
0.031

MOM

0.026

0.0
79

Reported method (1)

2.39

MOM

0.1–10 10–100

0.1–10

MOM
IND
2.67

Kinetex 1.7 μm HILIC 100A
column (100 × 2.1 mm, 1.7 μm
particle size)

Gradient

7

Reported method
[12]

MOM

233 nmMOM

0.092

Reported method (2)4.32

220 nm
MOM

7.29

IND
0.1

0.082

Reported method (3)MOM

50–300 1–150

IND

MOM

–

–

–

–

–

IND
0.03

MOM

0.0
02

Proposed spectrophotometricmethod

0.202

0.227

IND

1–30

IND

Proposed Spectroscopic
methods

0.5–100 1.1–32

IND

Methanol: 0.1% glacial acetic acid Methanol: 0.1% formic acid
(40:60, v/v), then methanol: 0.1% in water (20:80, v/v), then metha‑
glacial acetic acid (85:15, v/v)
nol: 0.1% formic acid in water(80:20, v/v)

Inertsil ODS C18 column(250 × 4.6 mm, 5 µm)

Gradient

10

Reported method[11]

10–100 50–300

IND

210 nm259 nmWavelength
IND

Methanol: micellar reagent(191.49 mM) and (10.00 mM)
potassium dihydrogen phos‑
phate buffer (22.50: 77.50 v/v),

Acetonitrile and acidified watercontaining 0.025% SDS as ion
pair reagent (50: 50% v/v)

Mobile phase

Linearity range (μg/mL) MOM

Shim-pack cyano column
Symmetry C18 column(150 mm × 4.6 mm; 5 μm particle (100 mm × 4.6 mm × 3.5 μm)
size)

Stationary phase8
Isocratic4

Isocratic

Run time (minute)

Reported method[10]Type of elution

Proposed IPC method

Parameter

Table 2 Comparative study of the proposed and reported methods

El‑Masry et al. BMC Chemistry(2024) 18:143
Page 11 of 15

RGB 12 algorithm

BAGI toolParameter

Table 2 (continued)

Proposed IPC method

Reported method[10]

Reported method[11]

Reported method[12]

Proposed Spectroscopic
methods

El‑Masry et al. BMC Chemistry(2024) 18:143
Page 12 of 15

El‑Masry et al. BMC Chemistry(2024) 18:143

just 0.035 kg ­CO2 eq versus 0.079–0.092 kg ­CO2 eq
for reported techniques. This over 50% reduction
reflects excellent environmental performance. Similarly, the spectrophotometry method showed the lowest emissions of 0.002 kg ­CO2 eq per sample analyzed.
This substantial decrease relative to prior reports is
owed to the very short analysis times cutting electricity demands combined with the simplified protocols
replacing organic solvents with water and ion pairing
agents markedly decreased related emissions. In summary, carbon footprint analysis confirmed the favorable
green credentials of the developed methods, with lower
greenhouse gas emissions demonstrating their positive
sustainability profile. The quantitative results reinforce
conclusions from ComplexGAPI and AGREE-based
evaluations regarding the techniques’ eco-friendly
nature.
Assessment of methods’ blueness based on BAGI criteria

BAGI tool enables quantitative evaluation of analytical methods’ functionality and real-world applicability(blueness) [35]. BAGI allows scoring across 10 practical criteria: analysis type, number of analytes detected,
instrumentation requirements, sample throughput,
preparation needs, analysis rate, reagents/materials consumed, preconcentration requirements, automation
potential, and sample quantity needed. Each parameter is graded from 1 (worst) to 10 (best), as shown in
Table S4. The composite BAGI index is computed as thegeometric [35]. The developed IPC and spectrophotometry methods obtained high BAGI scores of 87.5 and 90
respectively, confirming excellent practicality. The rapid
analysis times below 4 min for both drugs using simple
equipment and procedures offer significant real-world
advantages. Moreover, the eco-friendly ion pair reagent
minimizes material inputs and waste generation, echoing green analytical principles within an applied context.
Hence, the BAGI assessment verifies the outstanding
blueness of the techniques as implementable alternatives to replace conventional methods. However, since
BAGI specifically targets functional criteria, it does notprovide a holistic sustainability measure. Therefore, we
additionally employed the RGB12 algorithm to evaluate
the composite analytical sustainability considering greenness, performance, and practicality. The consistently highscores across tools verify this method’s merit as an implementable green chemistry alternative.
Evaluation of the methods’ whiteness profile

While tools like ComplexGAPI, AGREE, and BAGI provide assessments of specific sustainability aspects, theRGB12 algorithm enables quantitative evaluation of thecomposite analytical sustainability through whiteness

Page 13 of 15

measurement [36, 37]. RGB12 comprises 12 distinct
components divided into red, green, and blue groups
addressing parameters related to analytical validation,eco-friendliness, and practical feasibility respectively. The
green group (G1–G4) focuses on important parameterssuch as toxicity, excessive waste and reagent utilization,energy utilization, and the immediate effects on animals,
humans, and genetically modified organisms. The group
denoted by the color red (R1–R4) conducts an assessment of validation criteria, encompassing the extent ofapplicability, the LOD, LOQ, precision, and accuracy.
The cluster denoted as the blue group, comprising B1 to
B4, evaluates the affordability, time-based efficiency, as
well as practical and financial prerequisites. By applying the RGB algorithm, the whiteness value, which represents the method’s compliance with the ideas of WAC,
is estimated by combining the scores obtained for eachof the three-color groups. As shown in Table 2 and Fig.
S11, the developed methods attained excellent whiteness values of 88.1 (IPC) and 89.8 (Spectrophotometry).
This significantly exceeds reported techniques, confirming superior alignment with sustainability principles like
waste minimization, energy efficiency, precision, affordability, and practicality. The high scores reinforce conclusions from prior focused Greenness (ComplexGAPI,AGREE) and Blueness (BAGI) evaluations regarding thefavorable environmental and functional profile. Additionally, the outstanding red metric performance verifies excellent analytical validation. In contrast to othernarrowly-focused tools, RGB12 delivers a big-picture
quantification of sustainability. Using RGB12 alongsidespecialized gauges for greenness, blueness provided arobust, multidimensional assessment that avoids biases
from overemphasizing any single aspect like cost or precision. This systems-thinking approach applying multiple
complementary evaluation tools enables a comprehensive, unbiased assessment of alignment with sustainable
analytical chemistry ideals. The consistently high scoresvalidate the developed methods as greener, cost-effective
alternatives with comparable performance for wide-ranging applications.

Conclusion
The work presented in this study successfully developed
and validated three novel analytical approaches including
IPC and two spectrophotometric techniques for rapid,
simultaneous quantification of the anti-COPD drug combination—IND and MOM in pure and capsule preparations. Methods optimization and thorough validation as
per ICH guidelines demonstrated excellent sensitivity,
precision, accuracy, and linearity while analysis of labprepared mixtures and pharmaceutical capsules proved
specificity and reliability of results for routine quality

El‑Masry et al. BMC Chemistry(2024) 18:143

control. The proposed methods offer numerous advantages over earlier reports including straightforward protocols without elaborate extraction, and fractionation
steps; inexpensive reagents and equipment; eco-friendly
excluding toxic organics; very rapid analysis times; andexcellent detection limits aligning with therapeutic concentrations administered by oral inhalation. Additionally,
the developed approaches excel regarding waste minimization, cost-effectiveness, safety, and energy efficiency
thereby excellently addressing green analytical and sustainable white analytical chemistry goals. Holistic sustainability assessment using tools like ComplexGAPI,AGREE, carbon footprint, BAGI, and RGB12 verified
outstanding greenness, blueness, and whiteness metrics
alongside analytical reliability and practical functionality. Thus, this work contributes to a sustainable future foranalytical science by exemplifying benign practices thatreconcile environmental and economic aspirations.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​024-​01242-y.
Supplementary Material 1.
Author contributions
Amal A. El-Masry and Yomna A. Salem: carried out sample preparation, analy‑
sis, and writing the draft of the manuscript. Ahmed Emad F. Abbas: writing thedraft of the manuscript and approving the final manuscript.
Funding
Open access funding is provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB). This work wasn’t funded by any 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.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors state that they are not aware of any financial or interpersonal
issues that might have affected the study that was the subject of this work.
Author details
1
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura Uni‑
versity, Mansoura 35516, Egypt. 2 Analytical Chemistry Department, Facultyof Pharmacy, October 6 University, 6 October City 12585, Giza, Egypt. 3 Depart‑
ment of Pharmaceutical Chemistry, Faculty of Pharmacy, Sinai UniversityKantara Branch, Ismailia 341636, Egypt.
Received: 23 April 2024 Accepted: 8 July 2024

Page 14 of 15

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