Ali et al. BMC Chemistry(2024) 18:157
https://doi.org/10.1186/s13065-024-01265-5

BMC Chemistry

RESEARCH

Open Access

Chromatographic fingerprintingof ipratropium and fenoterol in their novel coformulated inhaler treating major respiratory
disorders; application to delivered dose
uniformity testing along with greenness
and whiteness assessment
Salma N. Ali1, Samah S. Saad1, Ahmed S. Fayed2 and Hoda M. Marzouk2*
Abstract
Ipratropium bromide (IPR) and fenoterol hydrobromide (FEN) have recently been combined in a promisinginhaler to treat two prevalent inflammatory illnesses of the airways: bronchial asthma and chronic obstructive
pulmonary disease (COPD). The necessity for a single, sensitive, and trustworthy analytical approach to cover
the diverse and necessary tests of in-vitro and in-vivo studies is greatly grown with the rising production of
new fixed combinations. Two novel, selective and environmentally friendly LC techniques were developed inorder to guarantee precise measurement of IPR and FEN in their challenging formulation. The initial techniqueinvolved high-performance thin-layer chromatography (HPTLC) in conjunction with densitometric quantification.
Chromatographic separation was attained on HPTLC plates utilizing ethyl acetate - ethanol - acetic acid
(5.0:5.0:0.1, by volume) as a developing system. Densitometric quantification of the separated bands was carriedout at 220.0 nm over concentration ranges of 0.50–15.0 µg/band for IPR and 0.50–12.0 µg/band for FEN. Highperformance liquid chromatography (HPLC) paired with diode array detection (DAD) was the core of the second
technique. The optimized separation was achieved on a Zorbax SB C18 (150 × 4.6 mm, 5 μm) column with a
combination of 10.0 mM potassium dihydrogen orthophosphate, pH 5.0 ± 0.1, adjusted with o-phosphoric acid andmethanol (70:30, v/v) as the mobile phase and pumped at flow rate of 1.0 mL/min. The peaks were monitored at220.0 nm using diode array detection, achieving linearity range of 5.0–200.0 µg/mL for both drugs. The ICH criteria
have been verified and both methods have been confirmed to be valid, and successfully applied for assay the citeddrugs in the Atrovent® comp HFA metered dose inhaler as well as delivered dose uniformity testing of the final
product. Finally, whiteness appraisal and several state-of-the-art green evaluation metrics were applied to evaluate
the sustainability of the proposed methods. The suggested approaches produced promising results and are the first*Correspondence:
Hoda M. Marzouk
hodaallah.marzouk@pharma.cu.edu.eg
Full list of author information is available at the end of the article© The Author(s) 2024. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use,sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) andthe source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in thisarticle are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not includedin the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you willneed to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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simple and sustainable methodologies for the simultaneous quantification of both drugs in different real samples,
all of which strongly suggest their application in quality control laboratories.
Keywords AGREE tool, Chronic obstructive pulmonary disease, ComplexGAPI, Delivered dose uniformity, EHS tool,
Fenoterol, HPLC–DAD, HPTLC–densitometry, Ipratropium, White analytical chemistry

Introduction
Bronchial asthma and chronic obstructive pulmonary
disease (COPD) are the two greatest inflammatory diseases of the respiratory tract, and both are increasing
worldwide affecting around 600 million people [1]. Bronchodilator treatments given by inhalation are helpful forthe symptomatic relief of constriction of the airway inpatients with bronchial asthma or COPD in adults andchildren [2] As a result, by developing a novel medication
combination of ipratropium bromide (IPR) and fenoterolhydrobromide (FEN), the pharmaceutical industry has
honed its energy to control and manage the symptoms ofboth COPD and asthma disorders [3].
IPR, a bronchospasm-related medication approved by
the Food and Drug Administration (FDA) [4], is an anticholinergic drug [Fig. 1a]. FEN is an inhaled bronchodilator medication for asthma. It is a β2-adrenergic agonist[Fig. 1b] [5]. IPR and FEN should be used together rather
than separately for the treatment of acute severe asthma
and COPD due to their different mechanisms of action,according to earlier study findings [6–8]. The potentialof this combination to increase forced expiratory volume
in one second (FEV1) has been studied [9]. Additionally,
patients needed less duration using metered dose inhalers (MDI) and experienced effective bronchodilation[6, 10]. On the other hand, taking each drug at a lower
dose together could still have the same clinical effect,
while potentially decreases the side effects of each drug if
administered alone [11].
The literature provided only one HPLC method for
determining the drugs under study along with otherdrugs in various nebulizer solutions [6]. This reported
work, in contrast to the proposed research, did notinclude the analysis of the co-formulated inhaler understudy (Atrovent® comp HFA) or delivered dose uniformity
testing. Moreover, the guidelines for green chromatography, which protect the environment and ensure analyst
safety, were also overlooked. Another study presents an
HPLC system to study factors affecting the stability andperformance of IPR and FEN pressurized-metered dose
inhalers [12], however this procedure was not validated
or applicable to dosage form assay.
The priority given to various analytical procedures haschanged as a result of the global trend towards environmentally friendly analytical methods depending on how
seriously they fulfil the guidelines for green analyticalchemistry [13]. The two most widely used and adaptable techniques in the field of pharmaceutical analysis arehigh performance thin layer chromatography (HPTLC)
and high-performance liquid chromatography (HPLC).
Both of them provide an automated, quick, simple, andeconomical method to separate, identify, and quantify
challenging mixtures with great repeatability and resolution [14, 15]. Chromatographic techniques have furthermore made major advancements in green analyticalchemistry. This was achieved by several attempts thathave been undertaken to reduce energy consumption, theusage of hazardous solvents in the mobile phase’s composition and waste production per sample. They facilitatedthe simultaneous analysis of several analytes [16, 17].
The purpose of this research study was to develop
the first simple, precise, and economical HPTLC andHPLC-DAD methods for the simultaneous determination of IPR and FEN in their challenging dosage form.
The motivation behind this research was to align with theglobal trend towards sustainable chemistry and develop
a more eco-friendly and safer alternatives. The study also
intended to prove that using green analytical techniquesin chromatographic separation was achievable without
losing analysis parameters. Each of the methods wereeffectively applied to the metered dosage inhaler and validated according to the ICH guidelines. Following that,a comparative analysis of the stated methods was performed, and their greenness profiles were evaluated via
several tools; including environmental, health and safety(EHS) tool, Complex Green Analytical Procedure Index(ComplexGAPI), Analytical Greenness metric (AGREE)
and White analytical chemistry (WAC).

Experimental
Instruments
For HPTLC–densitometry

The stationary phase used for the chromatographic
separation was HPTLC aluminum sheets (20 × 10 cm)
precoated with silica gel 60 F254 (Merck, Darmstadt,
Germany). Using a 100-µL CAMAG micro-syringe anda CAMAG Linomat 5 autosampler (Muttenz, Switzerland), the samples were dispensed onto the plates. A
CAMAG TLC scanner (model 3 S/N 1302319), runningwith winCATS software (Muttenz, Switerland) was used
for scanning and densitometric analysis. With reflectancemeasuring mode and a 20.0 mm/s scanning speed, theslit dimension was set to 3.0 × 0.45 mm. The radiation
source used came from a deuterium lamp.

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Fig. 1 Chemical structures of (a) Ipratropium bromide (IPR) and (b) Fenoterol hydrobromide (FEN).

For HPLC–DAD

The HPLC equipment used in the experiment was an
Agilent® 1260 HPLC separations module (Milford, UnitedStates), which included an auto-sampler, degasser, quaternary pump, column compartment, and photodiodearray detector (DAD). Agilent® chemStation software was
employed to process and modify data. A Zorbax® SB C18
column (150 × 4.6 mm, 5 μm) from GL Sciences (Barcelona, Spain), was employed. The pH was adjusted using
a Jenway® pH meter (model 3510) from (Felsted, Essex,
UK).
Reagents and materials
Pure samples

Pure standard IPR and FEN were kindly provided by the
Global Napi Pharmaceuticals (GNP) Company (Al-Giza,
Egypt). According to their BP official methods [18], theirpotency was checked and found to be 99.40% ± 1.062 and99.40% ± 0.926 for IPR and FEN, respectively.
Pharmaceutical formulation

Atrovent ® comp HFA (Batch No. 104604), each metereddose is labeled to contain 20.0 µg IPR and 50.0 µg FEN,
manufactured by Boehringer Ingelheim. It was boughtfrom Egyptian drugstore.
Chemicals and solvents

Ethyl acetate, ethanol, and glacial acetic acid (Pioneer
Chemical Co., Giza, Egypt) were all used as analyticalgrade chemicals and solvents, Potassium dihydrogenorthophosphate (Sigma-Aldrich, Steinheim, Germany).
Also, methanol of HPLC -grade (Fisher Scientific, UK)
was used.

Ali et al. BMC Chemistry(2024) 18:157

Standard solutions

To prepare individual stock solutions with a concentration of 1.0 mg/mL of IPR and FEN, 10.0 mg of eachstandard material was precisely weighed and put into
separate 10-mL volumetric flasks. The materials weredissolved in methanol and then diluted with methanol.
By methanol dilution of the stock solutions, working
standard solutions were prepared to achieve a concentration of 200.0 µg/mL. It was found that stock solutions for
IPR and FEN can be stored stable for up to a week at 4°C
in the refrigerator with light protection.
Procedures
Chromatographic conditions

For HPTLC–densitometry Each sample was applied
separately on HPTLC aluminum plates (20 × 10 cm) using
a 100-µL micro-syringe with autosampler. The samples
were placed 10.0 mm from the plates’ bottom border andthe sides in bands that were 6.0 mm broad. To accomplish
the separation, 60.0 mL of the developing system, composed of ethyl acetate – ethanol - acetic acid in a ratioof 5.0:5.0:0.1, by volume, was placed within a binary glasschamber. This developing system was left in the chamber for saturation at room temperature (25.0 ± 2°C) for
30.0 min. After that, the plates were allowed to develop
vertically, reaching a distance of 8.0 cm in a linear ascending direction. Following the developing procedure, theplates were then removed and dried with air. They were
densitometrically scanned at a measuring wavelength of
220.0 nm. The scanning speed was set at 20.0 mm/s. Densitograms, and integrated peak areas were the results ofthe scanning process.
For HPLC–DAD Separation was performed at room
temperature using a Zorbax® SB C18 column (150 × 4.6 mm,5 μm). The mobile phase composed of two solvents: thefirst, is 10.0 mM potassium dihydrogen orthophosphatewith pH adjusted to 5.0 ± 0.1 by o-phosphoric acid, andthe other solvent is methanol in ratio of 70:30, v/v. Solvents were degassed in an ultrasonic bath for 10.0 min
and passed through a 0.45-µm Millipore membrane filter
before use. The mobile phase was pushed through the column at a rate of 1.0 mL/min during the isocratic elution
process. After filtration, samples were injected into the
HPLC system in 10-µL quantities using an autosampler.
The separated peaks were detected and quantified at awavelength of 220.0 nm.
Linearity

For HPTLC–densitometry Using the HPTLC system,bands were applied using accurately measured aliquots of
the relevant IPR (0.5, 1.0, 2.0, 5.0, 10.0 and 15.0 µL) andFEN (0.5, 2.0, 5.0, 8.0, 10.0 and 12.0 µL) stock standard

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solutions in the range of 0.5–15.0 and 0.5–12.0 µg/band,
respectively. The procedure was carried out under thepreviously described chromatographic conditions. Calibration curves were generated by collecting scanning profiles, to plot the average integrated peak area against therelevant drug concentration where polynomial regression
equations were computed.
For HPLC–DAD Using the mobile phase as a diluent,linearity ranges were determined through preparing serial
dilutions of each drug, separately, with concentration
ranges of 5.0–200.0 µg/mL for IPR and FEN. Then, after
injecting each of the prepared solutions in triplicates,
chromatogram for each sample was obtained. The chromatographic conditions that were previously mentioned
were used, and the peak areas were integrated. Following
that, calibration curves were constructed to establish thecorrelation between the average integrated peak areas andits respective concentrations, and linear regression equations were demonstrated.
Analysis of laboratory-prepared mixtures

Aliquot quantities from stock standard solutions of theinvestigated drugs have been mixed in various ratios,
both above and below the branded ratio labeled in the
dosage form. This was performed to construct synthetic
binary mixtures for analysis. The samples were dissolved
and diluted with methanol or mobile phase to the proper
concentrations in 10-mL measuring flasks, and then analyzed under the aforementioned chromatographic conditions applicable to both methods.
Application to pharmaceutical formulation

Accurately 2.0 mL of the metered dose inhaler solution (Atrovent® comp HFA), equivalent to 800.0 µg IPR
and 2000.0 µg FEN, were transferred in 10-mL volumetric flasks. They were mixed with methanol anddiluted to reach the final concentrations of 0.08 and0.20 mg/mL, respectively. Appropriate dilutions withmethanol or mobile phase were applied to obtain concentrations within the linearity ranges for each of thedetermined analytes. Both methods applied using thepreviously described chromatographic system. The
respective regression equation was used to calculate
the recovery percent (R%) and the concentrations of theinvestigated drugs. Furthermore, the standard addition
technique was applied. This involved spiking exact quantities of standard IPR and FEN to Atrovent® comp HFA
nebulizer solutions and completing the mixtures withmethanol or mobile phase. The samples were then analyzed as previously described.

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Testing the delivered dose uniformity

value above 0.6, while the other band of IPR did not moveaway from the baseline. Using ethyl acetate-ethanol system, poor separation of the investigated compounds andtailed peaks were shown. The experimental results were
not efficient. Furthermore, various small quantities ofacetic acid, formic acid, or aqueous ammonium hydroxide solution were evaluated as potential additives to the
ethyl acetate-ethanol system. Initial results with ammonia and formic acid showed poor resolution with tailed
peaks. The addition of small amounts of acetic acid toethyl acetate-ethanol system achieved the desired outcomes; the shape of peaks was improved, and peak tailing problem was solved, while resolution was satisfactory.
The use of ethyl acetate - ethanol - acetic acid with different ratios was investigated and adjusted to provide thehighest resolution of the binary mixture and the optimum chromatographic separation; the system of choice
was ethyl acetate-ethanol-acetic acid (5.0:5.0:0.1, by volume), Fig. 2a. Several wavelengths (210.0, 220.0, 250.0,
and 275.0 nm) were considered for densitometric measurements; the one with the highest sensitivity for eachcomponent with minimum noise was 220.0 nm.

In compliance with international guidelines [19, 20], theproposed HPLC-DAD method was additionally applied
to evaluate the delivered dose uniformity of the marketed
combined inhaler. IPR and FEN content in Atrovent®
comp HFA inhaler were determined using the same procedure as previously described, with the exception thatjust two actuations (two dosage unit) were applied to a
5-mL volumetric flask and diluted with mobile phase to
the flask mark. Two distinct groups were used for thisstudy from two different batches of metered dose inhalers (GP1; B.N. 104604, GP2; B.N. 204632), each groupcontained 10 units. For each group, the procedure wasrepeated ten times, each time with a new delivered dosage unit.

Results and discussion
In quality control laboratories, where efficiency and costare critical, the release of new pharmaceutical formulations requires the use of straightforward, precise, affordable, and quick analytical techniques. Developing a safer
methodology presents a challenge in striking a balance
between reducing toxicity and preserving the efficacy ofthe method, (i.e.) eco-friendly approaches have evolved
as an attractive strategy [21]. This study is a more environmentally friendly option compared to other complicated chromatographic procedures since it requires
few-to-no tedious sample preparations and considered to
be straightforward, automated, and highly reproducible.
The concurrent analysis of multiple samples simultaneously conserves environmental resources by drasticallyreducing the amount of energy and solvents used for eachsample [22, 23]. Thus, the goal of this work is to develop
the first chromatographic techniques that are simple,
selective, and eco-friendly for the simultaneous quantification of IPR and FEN in their challenging inhaler dosage
form.
Method development and optimization
For HPTLC–densitometry

The evaluation of several developing systems with diverse
compositions and ratios served as the foundation for the
method development. To avoid their hazardous nature
and adverse environmental effects, chloroform, toluene,
and benzene were excluded from the trials. Several trials were conducted to optimize the mobile phase composition and scanning wavelength with the aim to reach
optimum separation between the drugs under study
and establish symmetrical peaks with appropriate Rf
values. Different eco-friendly solvent systems, such as
ethyl acetate-ethanol and ethyl acetate- methanol, were
examined in different ratios (5:5, 4:6, 6:4, 7:3 and 8:2,v/v) for acceptable separation performance. For the ethyl
acetate-methanol system, FEN band displayed a high Rf

For HPLC–DAD

The method was developed to be eco-friendly by avoiding the use and generation of harmful chemicals, minimizing waste, and providing a quick analysis time. By
this way, routine determination of IPR and FEN determination could be achieved without causing harm to theenvironment. Primarily, mixtures of methanol-water andmethanol-acetate buffer were tested in different ratios
at different pH values (4.0–6.0); but the resolution waspoor. After that, different methanol-potassium dihydrogen orthophosphate buffer ratios (with several pH values, 3.0–7.0) were tested by applying isocratic elution forstudying the parameters influencing chromatographic
separation performance. A 10.0 mM potassium dihydrogen orthophosphate at pH 5.0 ± 0.1, and methanol wereused in different ratios to provide reasonable results.
It was observed that excellent separation was achieved
upon raising the buffer ratio. Several stationary phasecolumns, including C8 and C18 in different dimensions,were carefully examined. The tried C8 columns displayed
non-resolved solvent and FEN peaks. By substituting
the C18 column for the C8 column, the separation wasenhanced and the optimal resolution for the analytes wasachieved. The best resolution was obtained using Zorbax®
SB C18 column (150 × 4.6 mm, 5 μm). Different wavelengths (210.0, 220.0 and 275.0 nm) were considered for
DAD measurements. The UV-absorption spectra of IPR
and FEN were recorded in the range of 200.0–400.0 nm
and were utilized for choosing the optimum wavelengthfor detection, Fig. S1. The selection criterion favored
IPR because of being lower constituent in the dosage

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Fig. 2 (a) HPTLC densitogram of resolved mixture of IPR and FEN (6.0 µg/band, each) and (b) HPLC-DAD chromatogram of resolved mixture of IPR andFEN (70.0 µg/mL, each)form ratio, which led to wavelength choice of 220.0 nm.
Additionally, the IPR spectrum shows low absorptivity athigher wavelengths. Furthermore, FEN, the synchronized
drug, exhibit high absorbance at the selected wavelength,
enabling the highest level of sensitivity for both drugs.
Optimal separation of the binary mixture was achieved
using an isocratic mobile phase composed of 10.0 mM
potassium dihydrogen orthophosphate (pH 5.0 ± 0.1,
adjusted by adding o-phosphoric acid) and methanol(70:30, v/v) with flow at a rate of 1.0 mL/min. The column
used for this method was Zorbax® SB C18 (150 × 4.6 mm,5 μm). It provided a satisfactory separation of the two
peaks in a short run time within 7 min. It demonstrated
high sensitivity for the investigated substances. Figure 2b

shows a chromatogram with the separated peaks withacceptable resolution and an appropriate analysis time.
System suitability parameters

System suitability parameters were monitored to ensure
the performance of the two optimized chromatographic
systems. For the HPTLC-densitometry technique, they
included retardation factor, capacity factor, tailing factor, resolution and selectivity [24], that were calculated
and provided satisfactory results for both drugs. Suitability parameters were also calculated for the HPLCDAD method. The acquired outcomes match up withthe acceptance values [25, 26] proving that the proposed

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Table 1 System suitability parameters of the proposed HPTLC–
densitometric and HPLC–DAD methods for the determination of
Ipratropium and Fenoterol

Table 2 Method validation parameters for determination of
Ipratropium and Fenoterol by proposed chromatographic
methods

MethodMethod Parameter

HPTLC-densitometry

MethodHPLC-DAD

Parameters

IPR

FEN

Retardation
factor (Rf)±
0.02aCapacity factor(k’)bSelectivity factor (α)cResolution (Rs)dTailing factor(T)eParameters

0.17

0.83

4.88

0.20

Retention time(tR) (min ± 0.1)
Selectivity factor (α)cResolution (Rs)dTailing factor(T)eColumn efficiency (N)f
Height equivalent to theoretical plate (mm/plate)g

Reference
value
[24]

24.40

α>1

14.88
0.83 1.00

Rs > 1.5
T≤2

FEN

IPR

Reference
value
[26]

2.89

5.56
α>1

2.86
3.43
1.10

1.00

Rs > 2T≤2HPTLC–densitometry
IPR
FEN
Linearity Range
0.50–15.0 0.50–12.0µg/band µg/band
Regression equation parameters
Slope (b)a----Coefficient 1 (b1)b-9.36-94.86
Coefficient 2 (b2)b670.272141.50
Intercept (a)a, b
162.17217.83
Correlation coefficient (r) 0.9999
0.9999Accuracy (mean ± SD)c
100.1399.15± 1.608± 1.215
Precision (±%RSD)- Repeatabilityd
0.786
0.671
- Intermediate precisione 0.944
0.838
Specificity (mean ±%RSD) 100.15
100.74± 1.185± 0.945
LODf
0.145
0.151
LOQf
0.439
0.459
Robustness (RSD%)g
1.105
1.820

HPLC-DAD
IPR
FEN
5.0-200.0 5.0-200.0µg/mL µg/mL
4.273-----5.899
0.999999.85± 1.218

20.418----11.208
0.9999100.10± 0.786

0.385
0.564
99.95
± 1.214
1.593
4.828
1.294

0.488
0.678
101.07± 0.239
1.515
4.591
0.697

a

Regression equation for HPLC: A = a + bc, where ‘A’ is the average peak area and‘c’ is the concentration (µg/mL)b2216 3555.45 N > 2000
0.067 0.042

aRetardation factor (Rf ) = distance traveled by the analyte/distance traveled by
the solvent frontbCapacity factor (k’); k’ = (1 – Rf )/Rf for HPTLC and k´ = (tR - t0) / t0 for HPLC.

c

Selectivity (α) = k’2/k’1, calculated for each of two successive peaksdResolution(Rs); Rs = Rf2 – Rf1/0.5 (w1 + w2), where Rf is the retardation factor andw is the peak width calculated for each of two successive peaks for HPTLC and
Rs = [2 (tR2 - tR1)] / (W1 + W2) for HPLC.
e

T = W0.05/2f, where W0.05 is the width of the peak at 5% height and f is the
distance from peak maximum to the leading edge of peak

Coefficients 1 and 2 are the coefficients of X2 and X, respectively. Following apolynomial regression: A = b1 × 2 + b2x + a, where ‘A’ is the average peak area, ‘c’is the concentration (µg/band), ‘b1’ and ‘b2’ are coefficients 1 and 2, respectivelyand ‘a’ is the interceptcAccuracy [average of five different concentrations of three replicates each(n = 15)]d

Intra-day precision [average of three different concentrations of three
replicates each (n = 9) within the same day]eInter-day precision [average of three different concentration of three replicateseach (n = 9) repeated on three successive days]f

LOD and LOQ are calculated from the standard deviation (SD) of they-intercepts and the slope of calibration curve (S) as follows: LOD = 3.3 × SD / S
and LOQ = 10 × SD / Sg

For HPTLC: average of the change in ethyl acetate ratio (± 1%), developing
distance (± 0.5 cm) and scanning wavelength (± 1 nm). For HPLC: average forthe change in phosphate buffer ratio (± 1%), flow rate (± 0.1 mL/min) and buffer
pH (± 0.1)

f

N = 16 (tR / w)2, where w is the peak widthg

HETP = L /N, where L is the column length (mm)method functioned well. Results obtained and data forboth methods are tabulated in Table 1.
Method validation

The ICH guidelines for the validation of analytical procedures [27] were implemented to evaluate the proposed
chromatographic methods’ validation criteria.
Linearity and concentration ranges

For HPTLC-densitometric method, calibration curves
were produced with polynomial regression equation,
displaying the correlation between the integrated peakarea and the corresponding concentrations within theranges of 0.5–15.0 and 0.5–12.0 µg/band of IPR and
FEN, respectively, Fig. S2. While the proposed HPLCDAD method possessed linear correlations in a rangeof 5.0–200.0 µg/mL for both drugs. Table 2 displays thecomplete parameters of the regression equations for eachmethod.
Accuracy and precision

Using the optimal chromatographic conditions, fiveindividual pure samples of IPR and FEN were examined to evaluate the accuracy of the suggested methods.
The regression equation relevant to each drug was used
to calculate its concentrations. As displayed in Table 2,
the accuracy of the proposed methods was validated byachieving the acceptable mean percentage recoveries.

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The assay of three various concentrations of each drug
was repeated three times on the same day and three consecutive days to assess the intra- and inter-day precisions,
respectively. After calculating the relative standard deviation (%), outcomes are shown in Table 2.
Selectivity and specificity

To evaluate method selectivity for both methods, laboratory binary mixtures for IPR and FEN were preparedin different concentrations within the aforementioned
linearity ranges. These mixtures had different ratios thatwere both above and below the claimed label of the dosage forms. Using the previously defined methods, several
laboratory admixtures were quantified. Table 2 gives animpression about the analysis results, including mean
percentage recoveries and percentage relative standard
deviation (RSD%) values. Detailed results are displayedin Table S1.These results point out satisfactory selectivity.
The complete separation of the binary mixture containing the two drugs under study, IPR and FEN, confirmed
the specificity of the two proposed methods, Fig. 2. Furthermore, no additional peaks or interferences from thecommon excipients or additives in the dosage form wereobserved, Fig. S3. In order to assess the purity of theresolved peaks from any co-eluting interferents, the work
also made effective use of DAD for recording UV absorption spectra at multiple points across IPR and FEN peaks.
Fig. S4 illustrates how the peaks of the two drugs werecolored green, and all scan paths (represented by black
diamonds) fell within the green zone at 990 peak threshold values [28–30]. Peak purity factors for IPR and FEN
were 999.934 and 999.973, respectively. Furthermore,
specificity was verified by examining the blank sample
chromatogram, as shown in Fig. S5.

Limit of detection and quantification

The slope of calibration curves and the standard deviation of y-intercepts of the regression lines were used for
calculating LOD and LOQ, Table 2. The proposed methods exhibit good sensitivity, as demonstrated by the low
LOD and LOQ values obtained.
Robustness

The ethyl acetate ratio (± 1%), developing distance(± 0.5 cm), and scanning wavelength (± 1.0 nm) changes
were averaged for HPTLC, whereas the phosphate buffer ratio (± 1%), flow rate (± 0.1 mL/min), and buffer pH(± 0.1) changes were averaged for HPLC-DAD. All of theoutcomes fell within the allowable range. The robustnessof the proposed methods was confirmed by ensuring thatthe validation parameters were maintained within an
acceptable range and the pooled relative standard deviation (%) was less than 2%, Table 2.
Analysis of the pharmaceutical formulation (atrovent ®
comp HFA)The Atrovent® comp HFA metered dose inhaler was successfully analyzed using the proposed HPTLC and HPLC
systems, confirming the absence of excipient interference. IPR and FEN in their dosage forms have been
selected. Furthermore, insurance of the validity of the
proposed methods was shown through the applicationof a standard addition technique. The outcomes are displayed in Table 3.
Evaluation of the delivered dose uniformity

For the quality control study of the final product of themetered dose inhaler, delivered dose uniformity test wasapplied to Atrovent ® comp HFA metered dose inhaler

Table 3 Quantitative estimation of Ipratropium and Fenoterol in Atrovent® comp HFA inhaler solution and application of standard
addition technique
Pharmaceutical HPTLC-densitometry
formulation
Atrovent®comp Drug %Found ± SD*
HFA(Each metereddose is labeled
to contain20.0 µg IPR and
IPR
100.78 ± 1.003
50.0 µg FEN)
)B.N. 104604)

HPLC-DADStandard Addition Technique
ClaimedPure%Recovery(µg/band)addedof the pure(µg/band) addedamount*
1.6

0.8
1.6
3.2

Mean ± SD
FEN

100.03± 1.121

4

Mean ± SD*

Average of five determinations

2.0
4.0
8.0

100.9399.47
99.71
100.04± 0.78399.34
101.7399.74
100.27 ± 1.280

Drug %Found Standard Addition Technique± SD*
Claimed Pure
%Recovery(µg/mL) addedof the pure(µg/mL)
added
amount *IPR
100.3120
10.0
100.59± 1.533
20.0
98.90
40.0
99.27
Mean ± SD
99.59± 0.888
FEN 100.04
50
25.0
100.94± 0.84750.0
100.40100.0
98.75
Mean ± SD
100.03± 1.141

Ali et al. BMC Chemistry(2024) 18:157

Page 9 of 14[31]. Using the HPLC-DAD technique, ten units for eachgroup were individually analyzed in accordance with theinternational guidelines [19, 20], to track the uniformityof delivered dosage units. The following formula wasused to calculate the dosage form acceptance value (AV)for each group [32].
AV = |M − X̄ | + ks.
Where ‘AV’ represents the acceptance value, ‘M’ is thereference value which equals ‘X̄’ (if 98.5% ≤ X̄ ≤101.5%);
it may also be equal to 98.5% or 101.5% (if X̄ <98.5% or X̄
>101.5%), respectively, ‘X̄’ is the mean recovery percent
for the assayed ten dosage units (two actuations each), ‘k’is the acceptance constant which equals 2.4 for ten units,and ‘s’ is the standard deviation of the units. If the ‘AV’ ofthe 10 dose units is less than or equal to 15.0%, the dosage uniformity requirements are satisfied. The calculated‘AV’ values are presented in Table 4. As shown in Fig. 3,
the ‘AV’ values for each group analyzed by the proposed
HPLC method were less than the maximum allowed
acceptance value (L1) of 15, proving an acceptable levelof delivered dosage uniformity.
Greenness profile assessment

In 2000, the term “Green Analytical Chemistry” (GAC)
emerged to reduce the negative effects that methods ofanalysis have on the environment and human health [33].
It has become crucial to strike a compromise between
obtaining results of highest quality and reducing theenvironmental risks caused by methods of analysis. Dueto their potentially harmful effects on human health andthe environment, selecting a solvent is one of the most
Table 4 Results of delivered dose uniformity testing for
determining Ipratropium and Fenoterol in Atrovent® comp HFAusing the proposed HPLC method
Atrovent® comp
HFA meter dose
no.

1
2
3
4
5
6
7
8
9
10
Mean
SD
RSD%
AV*
*

Label claim (%)
Group 1
(Inhaler 1; B.N.
104604)
IPR
FEN
100.4298.85
97.37
103.8997.52
104.56100.76104.51100.60100.37103.20103.79101.38101.13102.6299.07
103.07102.65102.24103.49100.92102.23
2.09
2.20
2.07
2.15
5.01
6.03

Group 2
(Inhaler 2; B.N.
204632)
IPR
100.4999.73
103.41102.73100.6898.21
100.69101.55102.35105.45101.53
2.05
2.02
4.95

FEN
103.4897.78
103.48103.74104.18103.94103.61103.9099.76
103.33102.72
2.15
2.09
6.38

Acceptance value = |M − X ̄ | + 2.4 × SD with maximum allowed level (L1) is 15

important steps in developing a method. Ethyl acetate
and ethanol are recommended as green solvents by thesolvent sustainability guide, which also color-codes acetic acid and methanol yellow due to their limitation [34,35]. Additionally, the three tools listed below were used
to assess and confirm the level of greenness for the proposed methods, namely, the Environmental, Health,and Safety (EHS) tool, the Analytical Greenness metric(AGREE) and the Complementary Green Analytical Procedure Index (ComplexGAPI).
Environmental, Health, and Safety (EHS) tool

Koller [36] has developed the environmental, health,and safety (EHS) tool for the quantitative screening ofpotential solvent hazards. This regarded to be a helpful
tool for assessing different solvents using nine hazardous
categories organized into three sets; the lower the score(closer to 0), the more environmentally friendly the solvent would get [37]. As shown by the EHS graph in Fig. 4,
the solvents utilized in the proposed methods; ethyl acetate and ethanol in HPTLC, and methanol in HPLC areconsidered to be the greener solvent when compared toother commonly used solvents in HPTLC method, suchas chloroform and toluene, and the commonly used solvent in HPLC method, acetonitrile. Also, the EHS graphshows preference for the solvents used in proposed chromatographic methods in comparison to that used inreported HPLC [6] method.
Complex Green Analytical Procedure Index (ComplexGAPI)This is a new and simple tool that improves the original
GAPI metric. The pictogram for GAPI, with five pentagrams, is expanded by a hexagonal field at the bottom in
the ComplexGAPI metric. This field reflects how “green”
pre-analysis procedures are. ComplexGAPI deals with all
steps of the pre-analysis and analysis process. The modified tool uses a color scale, just like in GAPI, with twoor three levels of assessment for each step. From green
to yellow to red, the generated pictogram can be used
to assess and quantify the low, medium, and high environment impact for each step, respectively. The several
aspects of the described processes and analytical protocol are represented by different fields. If certain requirements are met, these fields are filled in green [38]. By
looking at the obtained pictograms in Fig. 5, HPTLC
pictogram is greener than the HPLC-DAD pictogram,and both show less red-shaded sections, indicating green
analytical methods. Upon comparing the proposed chromatographic methods with the published HPLC method[6], the green color of the proposed methods was more
prominent because the used solvents have the highest
level of greenness in addition to using less waste per sample analysis as shown in Fig. 5.The hexagonal field in theAli et al. BMC Chemistry(2024) 18:157

Page 10 of 14

Fig. 3 Calculated AV values of IPR and FEN for group 1 (inhaler 1; B.N. 104604) and group 2 (inhaler 2; B.N. 204632) in Atrovent® comp HFA oral inhaler.
Each group of 10 units was individually assayed. The dashed line indicates the requirements of the USP Pharmacopoeia (AV ≤ 15%).

bottom with no colors, due to all methods have no preanalysis processes.
Analytical GREEnness metric (AGREE)

It is an advanced software for evaluating greenness [39].
A fraction of one, ranging from zero to one, is the finalscore in AGREE. Twelve sections make up the automatically generated pictogram. The color of each section
ranges from deep green (1) to deep red (0). The centerof the circular pictogram contains the final score. Basicprinciples like inclusivity, simplicity, flexibility in input
and output clarity were taken into consideration [40].
Figure 5 presented that both proposed methods show
AGREE pictograms with only one red zone, which correspond to off-line sampling. The overall score shownin HPTLC pictogram (0.79) is higher than HPLC one(0.73). But in general, overall score for both proposed
chromatographic methods indicate the highest ecologicalcompatibility and lowest negative impacts of these methods when compared to the reported HPLC [6] methodwith overall score (0.58), respectively, as shown in Fig. 5.
White analytical chemistry (WAC)

The goal of the white analytical chemistry method is toprovide a distinct tool for applying sustainable development concepts in analytical chemistry [41–43]. These
principles are composed of three complementary sections, each of which is colored differently (red, green, or
blue). These sections evaluate distinct concepts related tothe analytical method. Together, the aforementioned colors combine to produce the white color of the method. In
these pillars, the analytical method efficacy is evaluatedin the red section, the environmental impact is evaluated in the green section, and the practical usefulness
and economic conditions are simply evaluated in theblue section. The WAC tool is also referred to as RGB 12

Ali et al. BMC Chemistry(2024) 18:157

Page 11 of 14

Fig. 4 EHS assessment for different solvents used in the two proposed chromatographic methods and reported method [6]depending on the number of rules involved. “White” isdefined by the WAC as a purpose-driven, well-matchedanalytical approach [44, 45]. The recommended chromatographic methods were investigated and objectively
compared with each other and with the HPLC reported
method [6]. Figure 5 reports the evaluation results andthe arithmetic mean values for each of the three bands R(%), G(%), and B(%) of the proposed method using the
WAC tool. The overall score of 97.9%, it was determinedthat the prescribed HPTLC-densitometry performed
better than the proposed HPLC-DAD, which came insecond place with 92.6%. Reported HPLC method camein third place with overall score of 68.5%, as displayedin Fig. 5. The enhanced analytical performance of the
proposed HPTLC-densitometry and HPLC-DAD methods over the published HPLC method for the assay ofthe marketed dosage form was illustrated by the RGB12
algorithm tool in Fig. 5. Additionally, the ability of HPLCDAD method to test delivered dose uniformity expands
its scope of application. Because of their ease of use andgreen credentials, the proposed methods were found to
be more functional and sustainable.

Statistical analysis and methods ‘evaluation

A statistical comparison was performed between the
results of the IPR and FEN analyses in pure forms as
obtained by the proposed methods, and the results
obtained from their approved potentiometric titrationand titrimetric methods, respectively [18]. The t-test andF-test results showed that there was not a significant difference in terms of accuracy and precision between the
proposed and official methods, Table S2.
The proposed methods outperform the previously published HPLC method in terms of applicability, cost-effectiveness, efficiency, and ecological sustainability whenit comes to the simultaneous assay of FEN and IPR in
their metered dose inhaler dosage form. Additionally, thedelivered dose uniformity testing was performed by the
proposed HPLC-DAD method to evaluate the final product’s quality control, as illustrated in Table S3.

Conclusion
This work provided a successful attempt to develop
new, selective and versatile HPTLC–densitometric and
HPLC–DAD methods for the simultaneous determination of IPR and FEN in their challenging pharmaceutical co-formulated inhaler treating major respiratory

Ali et al. BMC Chemistry(2024) 18:157

Page 12 of 14

Fig. 5 Greenness profile and whiteness assessment of the proposed (a) HPTLC-densitometry, (b) HPLC-DAD and (c) the reported HPLC [6] methods via
ComplexGAPI, AGREE and RGB12 whiteness tool

disorders. Moreover, the higher sensitivity features of thedeveloped HPLC-DAD method was exploited for testing
delivered dose uniformity of IPR and FEN in Atrovent ®
comp HFA metered-dose inhaler. The concept of sustainable development has recently gained power among
analytical laboratories and instrumental corporations. A
lot of attention was given to using safer and less harmful solvents. Sustainable dominance of suggested methods than the reported HPLC one was ascertained via
three user-friendly, trustworthy and up-to-date greenness tools. Additionally, the RGB 12 algorithm, involving
freely available Excel sheet, was introduced as a holistic
evaluation tool, confirming high adherence to the WAC
concept. Clearly, the suggested methods are considered
promising alternatives for more sustainable, minimal
sample preparation, speedier, and cost saving, for drug
assaying in quality control laboratories while maintaining
the accuracy, sensitivity, selectivity, and precision of theanalytical determinations.

Supplementary Information

The online version contains supplementary material available at https://doi.
org/10.1186/s13065-024-01265-5.

Supplementary Material 1
Acknowledgements
Not applicable.
Author contributions
Salma N. Ali; Methodology, Software, Validation, Formal analysis,
Investigation, Writing—original draft, Writing—review & editing. SamahS. Saad; Conceptualization, Methodology, Supervision, Writing—review &editing. Ahmed S. Fayed: Conceptualization, Methodology, Visualization,
Supervision, Writing—review & editing. Hoda M. Marzouk; Conceptualization,
Methodology, Investigations, Software, Validation, Supervision, Writing—review and editing.
Funding
Not applicable.
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB).
Data availability
All data generated or analysed during this study is provided within the
manuscript or supplementary information files.

Declarations
Ethics approval and consent to participate
Not applicable.

Ali et al. BMC Chemistry(2024) 18:157

Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Author details
1
Pharmaceutical Analytical Chemistry Department, College of
Pharmaceutical Sciences and Drug Manufacturing, Misr University for
Science & Technology, 6th of October City, Giza, Egypt
2
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy,
Cairo University, Kasr Al-Aini Street, Cairo 11562, Egypt
Received: 25 June 2024 / Accepted: 9 August 2024

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