(2024) 18:133
Ashraf et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01238-8

BMC Chemistry

RESEARCH

Open Access

Novel RP‑HPLC–DAD approachfor simultaneous determinationof chlorphenoxamine hydrochloride
and caffeine with their related substances
Ahmed Ashraf1*, Norhan Badr ElDin1, Yasmin Rostom1, Badr A. El‑Zeany1 and Ghada A. Sedik1

Abstract
Ensuring the quality control of active pharmaceutical ingredients is crucial for drug products being introduced
into the market. Even for established drugs, it is necessary to maintain a cutting-edge impurity control system. To
analyze caffeine and chlorphenoxamine hydrochloride in their binary mixture, as well as theophylline and chlor‑phenoxamine N-oxide as related substances, a reversed phase-high performance liquid chromatography combinedwith a diode array detector system was created. The chromatographic separation was conducted using a C18
X-select Waters® column. The mobile phase consisted of 20.0 mM potassium dihydrogen phosphate modified
to pH 3 with o-phosphoric acid and methanol. A gradient elution program was adopted at a flow rate of 1.3 mL/
min and detected at a wavelength of 222 nm. The present methodology demonstrates a concentration ranging
from 2–60, 1–80, 0.5–20 to 0.4–20 µg/mL for chlorphenoxamine hydrochloride, caffeine, chlorphenoxamine N-Oxide
and theophylline, respectively. Chlorphenoxamine N-Oxide, being an impurity of chlorphenoxamine was pre‑pared by refluxing intact drug with 5% ­H2O2 for 24 h at 100 °C. One of the objectives of the analytical community
is to promote the adoption of green analysis methods, which involve the development of environmentally friendly
techniques. The levels of greenness and whiteness were evaluated using four specific tools: Eco-Scale System, GAPI,AGREE, and RGB tool. Furthermore, we have evaluated the greenness of the analytical method presented and com‑pared its performance and greenness to that of the approach described in the literature. In this study, results from CPX
and CAF analysis were compared to those obtained in a previous study. The result shows that there is no notable vari‑ation in precision and accuracy. The proposed method was validated in accordance with the requirements of ICH.
Keywords Caffeine, Chlorphenoxamine, Chlorphenoxamine N-Oxide, Green analytical chemistry, HPLC–DAD,
Theophylline*Correspondence:
Ahmed Ashraf
Ahmed.Ashraf@pharma.cu.edu.eg
1Analytical Chemistry Department, Faculty of Pharmacy, Cairo University,
Kasr El‑Aini Street, Cairo 11562, Egypt

Introduction
An allergic reaction is triggered when the immune
system responds to an external agent, such as pollen, specific types of food, insect bites or drugs [1, 2].
The range of symptoms associated with this condition spans from pruritus and respiratory irritation tourticaria and gastrointestinal distress [3]. Chlorphenoxamine hydrochloride (CPX); 2-[1-(4-chlorophenyl)1-phenylethoxy]-N,N-dimethylethanamine[4]; Fig. 1,© 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.

Ashraf et al. BMC Chemistry(2024) 18:133

Fig. 1 Chemical Structure of a Chlorphenoxamine HCl, b Caffeine, c
Chlorphenoxamine N-Oxide and d Theophylline

exhibits strong and sustained anti-histaminic andanticholinergic effects, making it a valuable treatment
option for allergic conditions that are linked to cholinergic activity. Furthermore, and based on the Off-label useof FDA-approved medications, it is noteworthy to highlight the paramount significance of chlorphenoxaminehydrochloride in the therapeutic intervention of Middle
East respiratory syndrome-related corona virus (MERSCOV), severe acute respiratory syndrome-related coronavirus (SARS-COV), Ebola virus (EBOV), and malaria
owing to its G protein-coupled receptor (GPCR) antagonistic properties [5–7]. Caffeine (CAF); 1,3,7-trimethylpurine-2,6-dione[8]; Fig. 1, is always combined with
CPX as its central stimulant effect nullifies the sedation action of CPX [9, 10]. Chlorphenoxamine N-Oxide(CPX N-Oxide); Fig. 1 is reported as CPX related substance [11, 12]. Theophylline (THP) is recognized as animpurity (related substance) of CAF, which results fromthe activity of cytochrome P450. This enzyme facilitates
the N-7 demethylation of CAF, leading to formation of
1,3-dimethylxanthine (THP); Fig. 1 [13]. In contemporary
times, the utilization of the Green analytical approachhas become imperative to mitigate the depletion of hazardous solvents or reagents, thereby diminishing theimpact of human activities on the environment. The
Green Analytical Chemistry (GAC) endeavors to transform the field of analytical chemistry towards achieving
environmental sustainability [14, 15]. This methodology
employs environmentally conscious sample preparation
techniques while also incorporating a concise time analysis. One of the objectives of utilizing the HPLC technique is to establish a state of balance between precision
and accuracy of outcomes while minimizing potential

Page 2 of 11

environmental risks [16], in accordance with the guidelines set forth by GAC [17]. Various analytical techniques
have been employed for the analysis of the proposed
drugs in their dosage forms, including but not limited to
HPLC [18, 19], supporting information Table S-1, spectrophotometry [20–22], differential pulse voltammetric[23], potentiometry [24] and thin layer chromatographic
densitometric method [25]. These methods have beenthoroughly researched and applied in the discipline ofpharmaceutical analysis. There is no analytical method
for analyzing CPX and CAF concurrently in the presenceof CPX N-oxide and THP as related compounds. HPLC,
being a widely employed analytical technique is utilized
for both separation and quantification of drugs underinvestigation. The goal of this research is to create a lowcost, high-sensitivity HPLC method [26]. The assessmentof the level of environmental friendliness was conducted
through the utilization of various tools such as National
Environmental Method Index [27], Analytical Eco-Scale[28], Green Analytical Procedure Index tool [29], Analytical Greenness Metric Approach and Software [30, 31].

Experimental
Instrumentation and software

An Agilent HPLC–DAD (1260 infinite II series) equippedwith a quaternary pneumatic pumping system (modelG1311C), an online degasser, a manual injector (20-μL)
and a photodiode array detector (DAD) (model G1315D)
was used in the experiment. In our study, we employeda Waters X select CSH® ­C18 (3.5 μm, 4.6 × 150 mm)­C18 analytical column [32] To operate the apparatus,
the Open LAB CDS ChemStation® software (version
C.01.07) was used. Mobile phase and sample underwent
filtration using 0.45-μm membrane filters and 0.22-μm
disposable nylon syringe filters, respectively. Adjustmentof pH was done by using pH meter (model 6300; Jenway,
UK).
Reagents

HPLC grade solvents were used, including potassium
dihydrogen phosphate, methanol, hydrogen peroxide ando-phosphoric acid Sigma-Aldrich (Steinheim am Albuch,
Germany).
Samples
Pure samples

Pure standards of CPX and CAF were obtained from EIPICO Pharmaceuticals, 10th of Ramadan City, Egypt, withpurities of 100.06 ±0.47% and 99.43 ±0.60% according toreported method [18]. THP was procured from SigmaAldrich (USA) with a certificate of purity of ≥ 99% [33]Ashraf et al. BMC Chemistry(2024) 18:133

Allergex® Caffeine tablets were produced by EIPICO
Pharmaceuticals 10th of Ramadan City, Egypt. The tablet
was supposed to have 20 mg CPX and 50 mg CAF. The
source of acquisition was a local pharmacy in Egypt.
Pharmaceutical formulation

Preparation of CPX N‑Oxide solution

For CPX N-Oxide preparation, 100 mg of CPX was
refluxed with 50 mL of 5% ­H2O2 for 24 h at 100 °C. The
degradation process was monitored by TLC method
throughout the disappearance of the spot corresponding to the intact drug and appearance of a new spot
referring to the formation of the assumed degradation
product using methanol: glacial acetic acid: water (5: 3:
2, by volume) as a creating system. The plates have beeninspected using a UV light with a wavelength of 254 nm.
After full degradation, a complete evaporation of thesolution to get rid of ­H2O2 content, then, 10 mg of CPX
N-Oxide was relocated into a 10 mL volumetric flask andsubsequently diluted with methanol to attain a concentration of 1.0 mg/mL. The process of oxidative degradation was further monitored by HPLC method and mass
spectrometry was used for the purpose of elucidating thestructure of the degradation product.
Procedure
Chromatographic conditions

The process of chromatographic separation was conducted within a temperature-controlled environment at
a temperature of (25 ± 2 ºC), utilizing a reversed phase­C18, X select column CSH. The gradient elution technique was carried out using a 20 mM potassium dihydrogen phosphate modified with o-phosphoric acid (pH3.0 ± 0.2) as solvent (A) and methanol as solvent (B). The
gradient has been established using three steps: initially, aratio of 70:30 v/v was maintained for the first 0–1.5 min,followed by a transition to a ratio of 20:80 v/v through thesubsequent 1.5–3 min. This ratio was then maintained
until the end of the run, from 3 to 10 min. Prior to usage,
the mobile phase was filtered through a 47 mm filter witha pore size of 0.45μm and was subjected to degassing
for 15 min in an ultrasonic bath. With the detection of
UV at 222 nm, a consistent flow rate of 1.3 mL/min was
obtained. After 10 min, the mobile phase was gradually
adjusted back to a ratio of 70:30 v/v, and then the mobile
phase was run until a stable baseline was achieved andthe pressure had reached a steady state.
Calibration curves construction

The working standard solutions of CPX, CAF, CPX
N-Oxide and THP (100 µg/mL) were prepared throughthe process of dilution using the corresponding stock

Page 3 of 11

solutions (1.0 mg/mL). Then, the linearity of each analyte
was assessed through serial dilution of working standard
solutions, resulting in a concentration range of, (2–60 µg/mL) for CPX, (1–80 µg/mL) for CAF, (0.5–20 µg/mL)
for CPX N-Oxide and (0.4–20 µg/mL) for THP with theused mobile phase as solvents. The solutions were subsequently passed through a 0.25 membrane and 20 µL ofeach solution were injected in triplets. The calibration
curves were created by comparing peak areas to the corresponding concentrations under the previously specified
chromatographic conditions.
Application to pharmaceutical formulation

Ten tablets had been balanced before being ground into
powder. An accurate portion of powder, equivalent to20 mg CPX and 50 mg CAF, was placed precisely into a
100 mL volumetric flask. To improve the drug extraction
process, 50 mL of methanol was introduced to the solution, which was then sonicated for 30 min. Methanol wasadded to the mark to complete the volume and the mixture was subsequently filtered using 0.25 µm membranefilter. Half milliliter of filtered solution was transferred
into a 10 mL volumetric flask and subsequently filled tothe mark with the mobile phase solution. The resulting
concentrations and the percentage of recoveries (R%) of
CPX and CAF were obtained using the regression equations. For application of the standard addition technique,
Allergex Caffeine powder was spiked with standard CPX(10, 20, 40 mg) and CAF (25, 50, 100 mg). The procedure
was completed as outlined in the analysis of the dosage
form.

Result and discussion
There has been a global push to promote environmentally
sustainable analytical methods that generate non-hazardous waste and minimize the production of harmful materials. The utilization of HPLC methodology allows for theefficient separation and quantification of components
within a brief timeframe, while using minimal quantities
of environmentally friendly solvents [34, 35]. The focus of
this study was to establish a precise and reliable analytical technique for detecting the studied analytes in theirprescribed dosage form, as well as in the presence theircorresponding related substances [36]. The suggested
approach mainly differs from the reported methods [18]by identifying related substances such as Chlorphenoxamine N-Oxide and Theophylline [37, 38]. It is crucial todetermine the presence of the caffeine impurity (Theophylline) as exceeding the permitted limit might lead toaccelerated heart rate or other cardiac rhythm disturbances, particularly in individuals with heart conditions[39].

Ashraf et al. BMC Chemistry(2024) 18:133

Characterization of oxidative degradation product of CPX

The degradation procedure, as mentioned in "Preparation of CPX N-Oxide solution" Sect., was monitored
through TLC technique. Various developing systems
were employed to achieve optimal separation between
CPX and its oxidative degradation product. An effective
separation was achieved utilizing a developing system
consisting of methanol, glacial acetic acid and water ina volumetric ratio of 5:3:2. The process of whole degradation was observed by tracking the removal of the drug
point located at ­Rf 0.52 and the emergence of a single
spot representing its oxidative degradation product at R­f0.75, as illustrated in Fig. 2 and supporting information
Figure S-1. This result was further confirmed by HPLC
and Mass spectroscopy using a mobile phase consistingof methanol: potassium dihydrogen phosphate modified
with o-phosphoric acid (pH 3) (80:20 v\v), starting with aconcentration of 60 µg/mL of CPX, Fig. 3a. The obtained
results indicate a reduction of approximately 50% in thepeak of the drug (­TR = 2.9 min), followed by the presence of a novel peak at 4.1 min, Fig. 3b. Following a 24
h degradation process, it was observed that only the oxidative product peak remained, while the CPX peak hadcompletely disappeared, as depicted in Fig. 3c. Moreover, the structure of CPX N-Oxide was illustrated by themass spectrum where a molecular ion peak was revealed
at m/z 353, Fig. 4, confirming the suggested pathway foroxidation as illustrated in Fig. 2.
Method optimization and development

The objective of the HPLC method design was to utilize
environmentally friendly chemicals and generate minimal hazardous waste while requiring a short analysis
time [40]. This approach was designed to routinely determine combinations including CPX and CAF, as well as
CPX N-Oxide and THP (as related compounds), without creating any environmental harm. Thus, two factors
were considered: solvents safety and the minimizationof the waste produced. Firstly, various combinations of
ethanol/water and methanol/water solutions were examined in isocratic separation mode, however the resolution
and separation results were unsatisfactory. Consequently,
potassium dihydrogen phosphate buffer instead of water

Page 4 of 11

was considered. Various ratios of ethanol and phosphatebuffer were tested at various pH levels (8 and 3), however,
the results indicated inadequate separation. Furthermore,
ethanol has a substantial disadvantage since the viscosity of ethanol/water mixtures is larger when compared tothat of methanol/water for the same eluotropic intensity
at ambient temperature [41]. High backpressures resulted
from such high viscosity. As a result, methanol was preferred over ethanol. Different ratios of phosphate buffer
and methanol were evaluated under various pH conditions via isocratic elution; however, this also resulted in
low resolution. Reasonable results were procured while
using methanol: potassium dihydrogen phosphate modified with o-phosphoric acid (pH 3). Even though raisingbuffer ratios CAF and THP revealed good separation,
CPX and CPX N-Oxide showed bad separation. On theother hand, increasing methanol ratio CPX and CPX
N-Oxide resulted in superior resolution, while CAF and
THP exhibited inadequate separation. Thus, gradient elution system was the best solution to gain better resolution
for all studied components. The drugs and their related
substances were effectively separated and quantified
using 20 mM potassium dihydrogen phosphate modified
to pH 3 with o-phosphoric acid (solvent A) and methanol (solvent B), using a gradient mode of 0 min to1.5 min(70:30 v/v) and 1.5–3 min (20:80 v/v) with a flow rate of
1.3 mL/min. At those adjusted conditions, peaks of eachcomponent were obtained with a short retention time
while maintaining satisfactory peak resolution. Furthermore, the suitability of the wavelength was evaluated bycustomizing the DAD detector at various wavelengths
to determine the optimal conditions yielding the highest
sensitivity for the analyzed components. The wavelengths
222 nm for CPX while 260 nm for CPX N-oxide, 272 nm
for the detection of CAF and THP were detected. HPLCchromatogram demonstrated that the retention time
for CPX was 5.018 ± 0.1 min, for CAF, it was 3.105 ± 0.1
min. While for CPX-N-Oxide, it was 6.182 ± 0.1 min andfor THP, it was 2.451 ± 0.1 min, Fig. 5. To verify that thesuggested approach was valid, a system suitability analysis was carried out. Six duplicate injections of the reference solution at 100% each were given for the CPX, CAF,
CPX N-Oxide and THP system suitability tests. Values ofFig. 2 Oxidative degradation pathway of chlorphenoxamine HCl using 5% ­H2O2 for 24h at 100˚ C to obtain chlorphenoxamine N-Oxide

Ashraf et al. BMC Chemistry(2024) 18:133

Page 5 of 11

Fig. 3 HPLC chromatograms during monitoring the CPX oxidative degradation at different time intervals starting with 60 µg/mL of CPX using
methanol-20 mM potassium dihydrogen phosphate modified with o-phosphoric acid pH 3 (80/20, v/v) at 222 nm. a Zero time degradation of CPX.
b After 12 hours degradation, peak of CPX N-Oxide has been observed. c After 24 degradation, peak of CPX has been disappered.

Ashraf et al. BMC Chemistry(2024) 18:133

Page 6 of 11

Fig. 4 The Mass Spectrum at negative mode showing molecular ion peak [M-2H] = 353 m/z for CPX N-Oxide

Fig. 5 HPLC Chromatogram of standard, CPX (20 µg/mL) ­(tR = 5.018), CAF (50 µg/mL) ­(tR = 3.105), CPX N-Oxide (10 µg/mL) (­ tR = 6.182) and THP(10 µg/mL) (­ tR = 2.45) ­C18 X-select Waters® column (3.5 µm, 4.6 mm × 150 mm) and a gradient elution of solvent A (20 mM potassium di hydrogen
phosphate adjusted to pH 3.0 with o-phosphoric acid) and solvent B (methanol) as a mobile phase at 222 nm

Ashraf et al. BMC Chemistry(2024) 18:133

Page 7 of 11

theoretical plates (≥ 2000), retention time (≤ 2.0%) andtailing factor (≤ 2.0) have been determined to be within
the standard acceptability guidelines for the system suitability parameters. Supporting information Table S-2
presents the calculations of the system suitability parameters. The acquired results were verified using the USP
Reference values [42].
Method validation

The proposed method validation was evaluated according to the guidelines set forth by ICH [43].
Linearity and range

Six concentrations were analyzed in triplicates within
specified ranges for each compound using RP-HPLC
method. CPX was examined in the range of 2–60 µg/mL,
CAF at 1–80 µg/mL, CPX N-Oxide at 0.5–20 µg/mL, and
THP at 0.4–20 µg/mL under specific chromatographic
conditions. There were linear correlations found between
the average peak areas and corresponding concentrations
as shown in Table 1.
Accuracy

procedures were accurate, supporting information Table
S-4.
Precision

Repeatability, three different concentrations were analyzed for CPX and CAF (5, 30, and 50 µg/mL) and for
CPX N-Oxide and THP (2, 6, 10 µg/mL). The results canbe found in Table 1, showing high precision with low values of percentage relative standard deviation (% RSD).
Inter-day precision was evaluated by replicating thesame three concentrations of the drugs on three successive days. The RSD values demonstrated limited variability, confirming that the data was reasonably consistent
Table 1.
Limit of detection and limit of quantification

The parameters LOD and LOQ were derived using theformulas LOD = 3.3 × SD/S and LOQ = 10 × SD/S, where
SD represents the standard deviation of the response(peak area) and S is the slope of the calibration curve,
Table 1.
Robustness

Five pure samples at concentrations of (7.00, 20.00, 40.00,
60.00, and 80.00 µg/mL) for CPX and CAF, and (3.00,
6.00, 12.00, 14.00, and 17.00 µg/mL) for CPX N-Oxide
and THP were examined in triplicate using RP-HPLC toverify the accuracy of the proposed methods. The percentage of recoveries were determined by utilizing theconcentrations derived from the appropriate regression
equations. The percentage recoveries from the suggested

Robustness is the capacity of a system to withstandminor variations in method parameters without being
affected. varying the pH by ± 0.2, adjusting the flow rateby ± 0.1 mL/min, and altering the wavelength by ± 2 nm.
The response was monitored by observing changes inpeak regions and calculating the %RSD. The results
indicated that small intentional changes in the tested
parameters did not affect the stability of the approaches,
supporting information Table S-5.

Table 1 Regression and validation Parameters of the proposed
HPLC -DAD method for determination of CPX and CAF and theirrelated substances in pure form

Analysis of the pharmaceutical formulation

Parameter

CPX

CAF

CPX N-Oxide

THP

Wavelength (nm)222 nm272 nm

260 nm272 nmLinearity
range (µg/mL)

2–60

1–80

0.5–20

0.4–20Intercept

14.567

12.694

11.4455.4162

Slope

55.70956.27555.48256.702

Correlation coefficient (r)0.99950.99970.99960.9993

Repeatabilitya

0.89

0.847

2.02

1.17

Intermediate ­precisionb

0.929

0.515

1.74

1.232

LODc(µg/mL)

0.59

0.312

0.15

0.12

LOQc(µg/mL)

1.79

0.945

0.43

0.33

Precision(± %RSD)a, bIntraday and interday precision [average three different concentrations of
three replicate each (n = 9) within same day and repeated on 3 successive days,
respectively]cof residuals
LOD and LOQ are calculated according to ICH, 3.3× SDslope
and

10× SD of residuals, respectively
slope

The co-formulation of CPX and CAF in Allergex® Caffeine tablets was effectively evaluated using the proposed
chromatographic method. The sample preparation was
conducted utilizing a single step extraction utilizing
methanol, indicating the removal of any intervention
from the tablet additives. The validity of this method anda standard addition technique were presented in supporting information Table S-3. The favorable results andminimal sample manipulation procedures also direct
focus towards the practicality of the suggested method as
environmentally friendly procedures for monitoring thequality of the analytes.
Statistical analysis

In this study, the results of CPX and CAF analysis werecompared to those previously achieved by a reported
one [18]. In supporting information Table S-4, it is foundthat the tabulated T and F values exceeded the calculated
ones. The suggested and reported methods demonstrated

Ashraf et al. BMC Chemistry(2024) 18:133

Page 8 of 11

no significant difference in terms of precision andaccuracy.
Assessment of the analytical method greenness profile.

The adoption of environmentally friendly analyticalmethods in the pharmaceutical industry, which replace
hazardous solvent-based methods, has gained momentum due to the increasing concern for environmental
protection [36, 44]. Within this context, assessing theenvironmental effects of various analytical methodologies in relation to their adherence to the guidelines ofgreen analytical chemistry (GAC) has been believed a
crucial undertaking [45]. Several evaluation tools for
GAC have been introduced, commonly referred to as"green metrics." These metrics serve to provide a quantitative or qualitative assessment of the level of environmental sustainability associated with each analyticalprocedure [46, 47]. The evaluation was carried out using
four different approaches.
a. Analytical eco-scale system
Eco-Scale grading system based on penalty scores
was devised as a partially quantitative way to measure how environmentally friendly the analyticalmethodology is [28]. The penalty scores are established
for all parameters in the analytical process which influences the optimum analysis, such as the quantity ofreagent, waste generated and energy exhaustion andare deducted from the ideal green technique base valueof 100. The Eco-Scale grading is located in Table 2. The
score of more than 75 attests to the method superior
practices.
b. Green analytical procedure index (GAPI)The combination of Eco-Scale and NEMI allows forthe assessment of the total sustainability of analyticalprocedures using a new instrument called GAPI [29].
A unique pictogram with five pentagrams to evaluate,
with the colors green, yellow and red signifying the low,
medium and high ecological influence for each step. The
GAPI tool provides information on sample preparation,
the influence of reagents on health and safety, waste
treatment and instrumentation. The GAPI pictogram isdisplayed in Table 2. Pictogram was created with more
green-shaded portions (7) than red-shaded ones (4), indicating a more green-focused analytical approach thanreported one.

Table 2 Greenness assessment of the proposed HPLC and reported HPLC method via different metrics
The proposed HPLC method
Analytical Eco-scale

GAPI Tool

Reagent
Methanol6

Phosphate Buffer

05% ­H2O2

4

Glacial acetic acid

4O-phosphoric acid4

Instruments
Energy1

Occupational hazard

0

Waste

6

Total penalty points

25

Total score

75

The reported HPLC method
Reagent
  Methanol

6  Acetonitrile

6

  Triethylamine

8

Instruments
Energy1

Occupational Hazard

0

Waste

6

Total Penalty points

27

Total score

73

AGREE Tool

RGB ModelAshraf et al. BMC Chemistry(2024) 18:133

c. Analytical greenness metric approach and software(AGREE)AGREE tool is the most recent consecrated universal
metric tool [30] that evaluates the degree of greenness of
the analytical approach based on the twelve principles,
supporting information Figure S-2. The tool output is aclock—like graph, assessment score is displayed in thecenter of the circular pictogram, with colors from deep
green to deep red. In this method the AGREE pictogramgave a score of 0.62, while for reported HPLC method
was 0.52 Table 2.
d. Assessment of whiteness of the proposed method
versus published method
The novel Red Green Blue (RGB) 12 algorithm withthree groups was acknowledged by Nowak et al. in 2021
[31]. Each group symbolizes a different color and contains
certain criteria that assess crucial facets of the analyticalmethod. Red group assesses the analytical effectivenessin terms of validation criteria, including scope of application, precision, accuracy and lowest LOD and LOQ. The
well-known GAC principles are assigned to the green
region and productivity factors including cost and timeeffectiveness, minimal practical requirements and operational simplicity are represented in the blue region. The
RGB 12 model is displayed as an Excel spreadsheet thatadhered to the WAC guidelines. The methodologies
are plainly evaluated in accordance with the 12 WAC
assumptions and the amount of sustainability as determined by the whiteness assessment is estimated. A wellbalanced analytical procedure that is appropriate for thesituation is referred to as "white" in the WAC approach.
The recommended approach was looked at and criticallycontrasted with the reported approach Table 2.

Conclusion
In this article, a newly developed and validated HPLC–DAD method was presented, offering several advantages
for the simultaneous determination of CPX, CAF and theirrespective related substances. The method is not only fast
and accurate, but also green and selective. Applying thismethod, it is possible to obtain highly sensitive and reliable
results, which can be crucial for a variety of analytical applications. Also, the greenness method was evaluated using
various assessment tools. These tools include Eco-Scalescoring, GAPI, and AGREE. The suggested method wasfound to have a minor environmental impact, as indicatedby the evaluation results. The application of this techniquefor determining CPX and CAF in tablets has been foundto be highly effective, as it is capable to accurately measure
the compound without being affected by any excipients.
The findings of this investigation shed light on the potential

Page 9 of 11

of CPX and CAF analysis as an alternative method for thequantification of the target compounds. To sum up, The
HPLC method proposed is a significant and advanced
approach for analyzing active pharmaceutical ingredients.
It is noteworthy for its eco-friendliness, uncomplicatednature with minimal data manipulation, reproducibility,
rapidity and precision.
Abbreviations
CPX	Chlorphenoxamine hydrochloride
CAF	Caffeine
DAD	Diode array detector
FDA	Food and drug administration
GAC​	Green analytical chemistry
MERS-COV	Middle East respiratory syndrome-related corona virus
ICH	International conference of harmonization
SARS-COV	Severe acute respiratory syndrome-related corona virus
THP	Theophylline

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​024-​01238-8.
Additional file 1.
Acknowledgements
Not applicable
Author contributions
Ahmed Ashraf: Writing the main manuscript text, methodology. Norhan Badr
ElDin: Figures preparation, analysis of all data. Yasmin Rostom& Ghada A. Sedik:
Conceptualization, writing review and editing. Badr A. El-Zeany: writing review
and editing. All the authors read, reviewed and approved the manuscript.
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB). Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB).
Availability of data and materials
The data used and/or analyzed during this study are available from the cor‑
responding author on a reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no known competing financial interests
or personal relationships that could have appeared to influence the work
reported in this paper.
Received: 3 January 2024 Accepted: 28 June 2024

Ashraf et al. BMC Chemistry(2024) 18:133

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