(2024) 18:131
El‑Maraghy et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01234-y

BMC Chemistry

RESEARCH

Open Access

Implementation of green‑assessed
nanotechnology and quality by design
approach for development of optical sensor
for determination of tobramycin in ophthalmic
formulations and spiked human plasma
Christine M. El‑Maraghy1*, Passant M. Medhat1, Rania M. Hathout2, Miriam F. Ayad3 and Nermine V. Fares3

Abstract
A fast eco-friendly colorimetric method was developed for the determination of Tobramycin in drug substance,
ophthalmic formulations, and spiked human plasma using silver nanoparticles optical sensor. Even though tobramy‑
cin is non-UV–visible absorbing, the developed method is based on measuring the absorbance quenching of silver
nanoparticles resulting from the interaction with tobramycin. Different factors affecting the absorbance intensity werestudied as; silver nanoparticle concentration, pH, buffer type, and reaction time using quality by design approach.
Validation of the proposed method was performed according to ICH guidelines and was found to be accurate, pre‑cise, and sensitive. The linearity range of tobramycin was 0.35–4.0 μg/mL. The optical sensor was successfully applied
for the determination of Tobramycin in ophthalmic formulations and spiked human plasma without pre-treatment.
Additionally, the binding between Tobramycin and PVP- capped silver nanoparticles was studied using moleculardocking software. The method was assessed and compared to colorimetric reported methods for the green character
using Green Analytical Procedure Index (GAPI) and Analytical GREEnness calculator (AGREE) tools and found to begreener.
Keywords Quality by design, Colorimetry, Sensor, Tobramycin, Green, Silver nanoparticles, AGREE, GAPI*Correspondence:
Christine M. El‑Maraghy
Christine_elmaraghy@hotmail.com; cmagued@msa.edu.eg
1Analytical Chemistry Department, Faculty of Pharmacy, October
University for Modern Sciences and Arts (MSA), 6th October City 11787,
Cairo, Egypt
2
Pharmaceutics and Industrial Pharmacy Department, Facultyof Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt
3
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy,
Ain Shams University, Abbassia, Cairo 11566, Egypt

Introduction
Tobramycin (TOBRA), Supplementary material Fig. S1,
is an aminoglycoside produced by Streptomyces Tenebrarius [1]. It is effective against gram-positive andgram-negative bacteria. TOBRA was widely used during COVID-19 pandemic to treat the secondary bacterial infection caused by viral pneumonia [2]. Moreover, it
was used to treat symptoms of conjunctivitis frequently
associated with COVID-19 patients [3]. The literature
reveals some spectroscopic [4, 5], and chromatographic[6, 7] methods for the determination of TOBRA but they
had the disadvantages of using the derivatization stepwhich is time-consuming, tedious, and not eco-friendly© 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‑Maraghy et al. BMC Chemistry(2024) 18:131

in addition, it lowers the method sensitivity. The analysis of TOBRA is challenging as it has no absorbance inthe UV/visible region, and it is very polar so has weakretention on the reversed-phase LC. For those mentioned
reasons, we aimed to develop a rapid green visual colorimetric method for the analysis of TOBRA, which does
not depend on its absorption nor utilize the derivatization process. Consequently, the absorbance quenching
method using nanoparticles was applied to achieve thisgoal. The principle of the absorbance quenching method
depends on decreasing the absorbance intensity of silver nanoparticles (AgNps) upon reaction with TOBRA
through hydrogen bond formation, resulting in aggregation of AgNps [8, 9], and hence, a color change occurred
which could be measured quantitatively. We chose the(AgNps) as they are inexpensive and have high extinction
for colorimetric detection [10], special optical and electronic properties [11], and they are widely used in biosensing, photonics, and in antimicrobial applications as
the manufacturing of biodegradable surgical sutures [12].
Additionally, new insights on the application of AgNps
were applied in drug delivery[13], glucose determination [14], DNA detection [15], analysis of drugs [16–18],
heavy metal ions [19, 20], anions [10, 21], and cations[22].
Few colorimetric methods were reported for the determination of TOBRA using sodium dodecyl sulfate (SDS)
capped -AgNps in milk and in exhaled breath condensate (EBC) [23–25], and using citrate capped -AgNp
decorated with TOBRA-specific aptamers for analysis
of TOBRA in milk [26], the comparison between thereported and proposed methods is shown in Supplementary material Table S1. In this work, the type of stabilizer
used for capping AgNPs is polyvinyl pyrrolidone (PVP);
as they have long polyvinyl chains that act as steric protective shields on the AgNps surface which make themstable and dispersed when compared to the SDS andcitrate capping agent [27]. Moreover, neither of the published methods applied the quality-by-design (QbD)
approach nor implemented the green chemistry principles for the colorimetric analysis of TOBRA in pharmaceutical preparation and human plasma. The (QbD)
approach enables us to choose the best reaction conditions using minimal number of trials to achieve maximum absorbance quenching and hence the optimum
method performance. Furthermore, none of these methods determined TOBRA in biological fluid. The green
character of the developed method was assessed andcompared to colorimetric reported methods by Green
Analytical Procedure Index (GAPI) [28] and Analytical
GREEnness calculator (AGREE) tools [29]. Additionally,
the interaction between the TOBRA and PVP capped
AgNPs (PVP-AgNPs) was assessed using molecular

Page 2 of 13

docking software. Thus, in this work, a fast, eco-friendly,and validated colorimetric method was developed for
determination of the non-chromophoric TOBRA basedon absorbance quenching of PVP-AgNPs in ophthalmic
formulations and spiked human plasma. Figure 1 shows aflow diagram that summarizes the steps of the proposed
method.

Experimental
Materials and reagents

TOBRA (purity: 100.74% ± 0.5), Al Andalous for pharmaceutical industries (Giza, Egypt), its purity was checkedby the colorimetric method [4]. PVP-AgNPs dark yellow,
water-soluble colloidal solution (particle size 40 nm ± 5nm) (Nanotech, Giza, Egypt). Orthophosphoric acid,acetic acid, and sodium hydroxide (LobaChemie, India).
Boric acid, sodium carbonate, and sodium bicarbonate(Oxford Laboratory reagent, India). Chloroform (Carlo
Erba reagents). Bi-distilled water was used throughout
the work. Human plasma was obtained from (VACSERA,
Cairo, Egypt) and stored at − 20 °C until use.
Instrumentation

A double beam spectrophotometer (UV-1800, ShimadzuJapan) connected to a PC with UV-probe 2.10 software.
Transmission electron microscopy (TEM) (JOEL JEM1400, USA). Sigma Centrifuge (model no. D-37520,
Germany). Jenway pH glass electrode (Model P14/BNC,
Stone, UK).
Design expert® software version 7.0 was used for experimental design. MOE® version 2014.0901 (Chemical
Computing Group Inc., Montreal, Canada) was utilized
in docking study. ChemDraw® Ultra version 10 (Cambridgesoft, Waltham, MA) and Chem3D® Ultra version
10 (Cambridgesoft, Waltham, MA) were utilized fordrawing the chemical structures.
SoftwareTobrin® ophthalmic solution (batch No. 1801933), 3000
μg TOBRA per 1 mL, EIPICO company and Tobrin®
ophthalmic ointment (batch No. 2010036), 3000 μg
TOBRA per 1 gm, EIPICO company.
Pharmaceutical formulations

Buffers preparation

Britton-Robinson buffer was prepared by mixing equalvolumes of acetic acid, boric acid and, phosphoric acidadjusted to the required pH by 0.2 M NaOH [30]. Carbonate buffer was prepared by mixing 0.1 M sodium carbonate and 0.1 M sodium bicarbonate, adjusted to therequired pH by 0.2 M HCL [31].

El‑Maraghy et al. BMC Chemistry(2024) 18:131

Page 3 of 13

Fig. 1 A flow diagram that describes the steps of the proposed method

Standard and working solutions

Stock solution of TOBRA was prepared (1000 μg/mL)using bi-distilled water. The working solution was prepared by dilution from the stock to reach a concentration of (50 μg/mL).
Procedure
Absorbance quenching produced by TOBRA‑PVP‑AgNPs
reaction

A solution of PVP-AgNPs of concentration (215.74 µg/mL) was prepared using a solvent mixture of BrittonRobinson buffer (pH 9) and bi-distilled water in a ratio(50:50 v/v) and its absorption spectrum was recordedat λmax = 415 nm using the same solvent mixture as a
blank. An aliquot of TOBRA was added to the previously prepared solution of PVP-AgNPs to obtain a final
concentration of 4 µg/mL TOBRA then the solution
was left to stand for 10 min and the absorbance wasmeasured at 415 nm. The reaction between the TOBRA
and the PVP-AgNPs resulted in a color change fromyellowish-brown to orange.

Exploring the mechanism of tobramycin absorbancequenching due to the used PVP‑capped silver
nanoparticles using molecular docking studies
Building PVP polymeric matrix

The PVP polymeric matrix constituting of 5 or
10 monomers was constructed from the polymer
SMILES;CC(N1CCCC1(=O))CC(N1CCCC1(=O))
CC(N1CCCC1(=O))CC(N1CCCC1(= O))
CC(N1CCCC1(=O))CC(N1CCCC1(=O))
CC(N1CCCC1(=O))CC(N1CCCC1(=O))
CC(N1CCCC1(=O))CC(N1CCCC1(=O))CC, utilizing
the chemical sketch tool of the RSCB protein data bank
(RCSB PDB: Chemical Sketch Tool).
After, the generated pdb files of the investigated polymer was imported to MOE® version 2014.0901, where
the energy minimization of PVP chemical structure was
performed using the MMFF94x forcefield simulation
tool found in the MOE software package [32].

El‑Maraghy et al. BMC Chemistry(2024) 18:131

Construction of the chemical structure of tobramycin
and its preparation for docking

The chemical structure of TOBRA was built using ChemDraw® Ultra version 10 from its isomeric SMILES thatwere obtained from PubChem [33]. The corresponding
Mol2 file (usually needed for docking experiments) was
obtained using Chem3D® Ultra version 10. The energy
minimization to obtain the most stable 3D configurationin space was performed utilizing the MM2 forcefield of
the same software [34].
Molecular docking studies

The molecular docking analysis was adopted using
MOE® version 2014.0901. Binding sites identification
was carried out using the MOE’s "Site finder" tool [35].
The "triangle matcher” as the placement method in thedocking technique [36] and the London score of the software was exploited to generate the free binding energies
results of the docking process.
Validation of the proposed method

The analytical method validation of the proposed method
was done according to International Council on Harmonization recommendations [37]. For the linearity,
aliquots of TOBRA were added to the solution of PVPAgNPs to obtain concentrations range (0.35–4.0 μg/mL) then the same procedure was followed. Calibration
curve was constructed by plotting (A⁰- A) versus the corresponding concentration of TOBRA and the regression
equation was calculated. Accuracy was assessed by measuring the average recoveries and SD of four concentrations of TOBRA (0.6, 0.7, 2.0 and 2.5 μg/mL) using thecorresponding regression equation. The intra-day precision (conducted in the same day) and inter-day precision(conducted in three successive days) were determined bymeasuring three concentrations of TOBRA (1, 2.5, and3.0 μg/mL) and the relative standard deviation was calculated for each concentration. Approaches based on the
SD of the intercept and the slope were used for determining the LOD and LOQ, where LOD = 3.3 × SD/slope and
LOQ = 10 × SD/slope.
Application to pharmaceutical preparations
Ophthalmic solution

1 mL of Tobrin® ophthalmic solution was transferred into
a 100 mL-volumetric flask and then the volume was completed with bi-distilled water (stock 1). Then, 150 μL weretransferred from (stock 1) into a 10 mL-volumetric flask
to obtain a final concentration of 0.45 μg/mL of TOBRA.
The absorbance quenching of PVP-AgNPs after the addition of the prepared TOBRA solution was measured. The
recovery was obtained from the regression equation. The

Page 4 of 13

standard addition technique was applied by adding different known concentrations of TOBRA to a known concentration of Tobrin® preparation.
0.06 gm of Tobrin® ophthalmic ointment was accuratelytransferred into a separating funnel, dissolved in 1.5 mL
chloroform, shaken vigorously for 1 min then extractedwith 3 cycles of 10 mL water. The three aqueous extracts
were mixed and diluted with bi-distilled water to reachthe final concentration of 1μg/mL TOBRA. Then thesame procedure was followed, and the standard addition
technique was applied.
Ophthalmic ointment

Application to human plasma

The ophthalmic route of TOBRA has some systemic
absorption; the mean peak serum level reaches 5 µg/mL[38] and consequently, it may cause side effects such as
headache and laryngospasm [39]. Therefore, a fast andsensitive method is required for its determination in
Human plasma. 1 mL plasma was spiked with different
aliquots of TOBRA transferred from the working solution and 2 mL of methanol was added for extraction. The
samples were mixed well and centrifuged at 8000 rpm
for 10 min. The supernatant was transferred into three
10 mL-volumetric flasks to prepare (0.5, 1, and 5 µg/mL)
TOBRA solutions, then analyzed as described.

Results and discussion
Being a non-chromophoric drug, the spectrophotometricanalysis of TOBRA is challenging. Spectrophotometricanalysis has many advantages, as it is a time and cost-saving, straightforward technique, which does not require alarge volume of solvent so it is considered a green alternative to the commonly applied chromatographic methods.
This work applied the absorbance quenching techniqueusing PVP-AgNPs to measure the TOBRA concentration
at a specific wavelength. There are two published works
for the colorimetric analysis of TOBRA [4, 5]. Both methods have higher LOQ than the proposed method (500 µg/
mL and 3 µg/mL, respectively versus 0.3 µg/mL) in addition they are less accurate and less precise which is due tothe derivatization step by the diphenylamine reagent in
the first method and vanillin in the second method while
applying high temperature (around 100 °C). Moreover,neither of the published methods applied the quality-bydesign approach nor implemented the green chemistry
principle for the spectrophotometric analysis of TOBRA
in pharmaceutical preparation and Human plasma, acomparison between the two colorimetric publishedmethods and the proposed method is shown in Supplementary material Table S2.

El‑Maraghy et al. BMC Chemistry(2024) 18:131

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Fig. 2 a UV–visible absorption spectrum of 215.74 μg/mL PVP-AgNPs (--------) and of PVP-AgNPs reaction with 4 μg/mL TOBRA (………​…………)
b TEM image of PVP-AgNPs

Characterization of PVP‑AgNPs

A solution of PVP-AgNPs was characterized by UV spectrophotometer and TEM. The spectrum showed that
PVP-AgNPs have SPR peak at 415 nm, which is characteristic for the silver nanoparticles [40, 41] (Fig. 2a). TEM
image showed that PVP-AgNPs are spherical in shape,

dispersed, and uniform in aqueous solution with particle
size 40 nm ± 5 nm (Fig. 2b).
The mechanism of absorbance quenching of PVP‑AgNPs

PVP-AgNPs form a yellow colloidal solution due toexcitation of surface Plasmon resonance (SPR) whichEl‑Maraghy et al. BMC Chemistry(2024) 18:131

gives maximum absorption at 415 nm. SPR is the collective oscillation of the conduction electrons on themetal surface when excited by a light of a specific wavelength [40]. The mechanism of PVP-AgNPs aggregation is due to hydrogen bond formation [8] between
the lone pair of electrons of the oxygen in the polyvinyl groups of the PVP-AgNPs and hydroxyl and aminogroups of TOBRA, Supplementary material Fig. S2. The
approach is based on the interaction between opposite charges of AgNPs and TOBRA as the amino andhydroxyl functional groups of TOBRA work as molecular bridge, starting the electrostatic coupling with theneighboring AgNPs. Consequently, an aggregationof the AgNPs occurs and the SPR band of the AgNPs
undergoes a hypochromic shift leading to a visible color
change from yellowish-brown to orange [9]. Thus, theproposed method is based on measuring the decrease
in SPR peak of PVP-AgNPs at 415 nm by increasing
the concentration of TOBRA, (Fig. 2a) and hence theabsorbance difference (absorbance quenching) between
the PVP-AgNPs solution and the TOBRA- PVP-AgNPs
solution was measured.

Page 6 of 13

Probing the tobramycin absorbance quenchingmechanism due to the used PVP‑capped silver
nanoparticles using molecular docking studies

Based on the molecular docking studies, TOBRA wasfound to successfully interact with PVP (ΔG =− 10.23 and− 8.19 kCal for the simulated 5-monomer and 10-monomer PVP, respectively). This was attributed to hydrogenbond formation between the lone pair of electrons of theoxygen in the polyvinyl groups of the PVP and hydroxyl
and amino groups of TOBRA, as demonstrated in Fig. 3
Preliminary studies
Effect of pH on the absorbance quenching

To select the pH for optimum sensitivity of TOBRA with
PVP-AgNPs, Britton -Robinson buffer solutions having different pH ranges (5–11) were prepared and tried.
The pH range (7–9) was selected for further optimization
by quality by design approach. This is because at pH ≤ 4,
the moderately increased ­H+ allows the formation of“bridges” between pyrrolidone groups and consequently
contracting the AgNPs capping or possibly interconnecting adjacent AgNPs resulting in an increased aggregation

Fig. 3 Molecular docking experiments of TOBRA on the capping agent; PVP: A and B represents the 3D docking results on the 5 and 10 monomeric
PVP matrix, respectively, while C and D represents the corresponding 2D images of the above panel. Tobramycin molecule is presented with balls
and sticks while the PVP matrix is presented with wires

El‑Maraghy et al. BMC Chemistry(2024) 18:131

rate with higher Zeta potential values [42]. This may
also be due to the neutralization of the PVP-AgNPs surface charges and/or absence/reduction of electrostatic
repulsion ultimately leading to an increase in the destabilization of the particles and increased particle aggregation [43, 44]. An Increase in pH (from 7 to 9) leads toan increase in the concentration of ­OH− thus increasing
electrostatic repulsion of PVP-AgNPs and thereby providing stabilization at alkaline pH which is translated as
decreased particle size with small hydrodynamic diameters and low zeta potential—a marker for high colloidal
stability [45]. Moreover, increasing the pH over 9, mightlead to the formation of ­Ag2O black precipitate and thus
negatively affecting the SPR peak intensity [27].
Effect of silver nanoparticle concentrationon the absorbance quenching

Different concentrations of PVP-AgNPs solutions ranging from (53.93–215.74 µg/mL) were studied. PVPAgNPs concentrations above 215.74 µg/mL were
excluded as they have absorbance values above 2 units.
Effect of reaction time on the absorbance quenching

After trying different time intervals for the reaction
between TOBRA and PVP-AgNPs, results revealed
that the reaction was completed in 5 min, where theabsorbance quenching reached its maximum value andremained stable for 30 min. consequently, the reaction
time was fixed at 5 min, Supplementary material Fig.
S3 shows the reaction time effect on the absorbancequenching.
Choice of the capping agent

Capping the AgNps with PVP has many advantages over
the SDS and citrate which are used as capping agents.
The advantages of PVP over the SDS capping agent; 1—
the former leads to the synthesis of more uniform particle
size, 2—PVP-capped AgNPs have superior antibacterial
and anti-fungal activities; which prolongs their shelf-life[46], 3—PVP-capping helps to stabilize the AgNPs byforming steric protective shields, allowing steric and electrostatic stabilization [17], and finally 4—PVP-capping
provides excellent physicochemical properties like solubility in both water and organic solvents, biocompatibility, and non-toxicity [47]. Comparing the PVP-capped
AgNPs to citrate-capped AgNPs; PVP-capped was foundto be more stable [48] and had smaller particle size which
improved the method sensitivity [49].
Optimization of conditions using Quality by Design
approach

Quality by Design provides substantial benefits in thefield of analytical chemistry, specifically by improving

Page 7 of 13

analytical methodologies and saving time and money.
A key benefit of QbD is the robustness it brings to analytical processes with the least number of experiments.
It guarantees the durability and constant performance ofanalytical procedures by precisely defining objectives andcomprehending the connections between critical procedure factors. Additionally, it improves the predictability,
reliability and effectiveness of method development, enabling faster refinement and adjustment of analytical procedures [50, 51].
The full factorial design ­(23) was used to determine theeffect of three factors; the type of buffer, pH and PVPAgNPs concentration to get the optimum conditions
for maximum absorbance quenching for the reaction
between TOBRA and PVP-AgNPs with the highest sensitivity. The design permitted to study the significance ofeach chosen factor and its interaction with the other factors. Two levels for each factor were studied: two typesof buffers (Britton-Robinson and carbonate buffers), pH(7 and 9) and PVP-AgNPs concentration (53.93 μg/mL
and 215.74 μg/mL), (Supplementary material Table S3).
To estimate the model coefficients, ANOVA with linear
model was applied. The results of 2
­ 3 design, are 20 trials,
including four center points, (Supplementary material
Table S4). The samples were measured by spectrophotometer at 415 nm.
Analysis of the results

The experimental design results shown in Table 1, indicate that the model fits the experimental data as thep-value is < 0.0001 and the F value is equal to 54.73. Buffer
type (factor B) and, PVP-AgNPs concentration (factorC) are the most significant factors (with p < 0.0001). The
model is linear and revealed that R2 is equal to 0.9746
which means that almost all values of the dependent andindependent variables lie on a straight line. Predicted andadjusted ­R2 are equal to 0.8997 and 0.9788, respectively,
which indicates that the model is significant and canpredict unplanned experiments. Moreover, the adequate
precision is greater than 4 which proves that this modelis adequate to navigate the space and the difference inresults is not random [52].
The studied factors and their interactions

The model adequacy checking was applied to explain
whether the fitted model provides the approximation to the true system. The significance of one factor
plots, (Fig. 4), indicates that the absorbance quenching
increases when using Britton-Robinson buffer (factorB), by increasing the PVP-AgNPs concentration (factorC) and by increasing the pH (factor A). (Supplementarymaterial Fig. S4a) showed the interaction between the
pH and the buffer while the PVP-AgNPs concentration

El‑Maraghy et al. BMC Chemistry(2024) 18:131

Page 8 of 13

Table 1 ANOVA results of the proposed full factorial model
SourceSum of squares

Degree of freedom

Mean squares

F value

P values

Model

2.460

7

0.350

54.73

< 0.0001a

A-pH

0.170

1

0.170

26.59

0.0004aB-Buffer type

0.370

1

0.370

57.92

< 0.0001a

C-PVP-AgNps Concentra‑
tion

1.290

1

1.290

201.01< 0.0001a

AB

0.072

1

0.072

11.28

0.0073a

AC

0.170

1

0.170

25.95

0.0005a

BC

0.300

1

0.300

46.05

< 0.0001a

ABC

0.048

1

0.048

7.55

0.0206a6.414 × ­10–3

Pure error

0.064

10

Cor total

2.560

19

a

Significant p < 0.05

Fig. 4 Significance of single factor a pH b Buffer Type and c PVP-AgNPs concentration on the absorbance quenching

was kept constant; maximum absorption quenching
was achieved at pH 9 with both buffers. (Supplementarymaterial Fig. S4b) showed the interaction between the pH
and the PVP-AgNPs concentration using Britton-Robinson buffer; the PVP-AgNPs of concentration (215.74
μg/mL) at pH 9 had higher absorbance quenching thanat pH 7 (Supplementary material Fig. S4c) showed theinteraction between buffer type and PVP-AgNPs concentration, while the pH was constant; Britton-Robinson

buffer showed higher absorbance quenching than Carbonate buffer. The pareto-chart in (Supplementary material Fig. S5) showed the influence of each factor and eachinteraction on the absorbance quenching; PVP-AgNPs
concentration and the buffer type were found to be themost significant factors. The Linear correlation plot of
predicted versus actual values showed that almost all
the points are close to the 45⁰ diagonal line which means
minimum deviation Supplementary material Fig. S6).

El‑Maraghy et al. BMC Chemistry(2024) 18:131

Page 9 of 13

Fig. 5. 3D-surface plot a, b and 2D-contour plot c, d showing the effect of pH and PVP-AgNPs concentration on the absorbance quenchingof PVP-AgNPs in presence of 4.00 µg/mL TOBRA in case of using Britton-Robinson buffer a, c and Carbonate buffer b, d

The 3D-surface plots (Fig. 5a, b) and two-dimensional(2D) contour plots (Fig. 5c, d) showed the interaction
between pH and PVP-AgNPs concentration and its
effect on the absorbance quenching value; as the PVPAgNPs concentration increases, the absorbance quenching increases regardless of the buffer type and pH value.
The blue region showed the experiments that resulted
in low absorbance quenching and the red region indicated the experiments of high absorbance quenching.
We can conclude that the highest absorbance quenching was achieved with PVP-AgNPs concentration rangeof (175.29–215.74 μg/mL) and Britton-Robinson buffer(Fig. 5c) gave higher absorbance quenching values (redregion) than Carbonate buffer (no red region) (Fig. 5d)at the same pH and PVP-AgNPs concentration values.
Therefore, the optimum conditions for our method were
PVP-AgNPs concentration of 215.74 μg/mL using Britton-Robinson buffer of pH = 9. The validity of the design
was proved by the correspondence between the experimental and the predicted results, which was found to be
99.95% at the optimum levels.

Validation of the proposed method

Calibration curves were constructed by plotting (A⁰–A)
versus the corresponding concentration. The regression
Table 2 Results of assay validation of Tobramycin by the
proposed method
Parameters

Results

Linearity (μg/mL)

0.35–4Slope

0.1903

Intercept

0.3339Correlation coefficient (r)0.9997abAccuracy (mean % ± SD)

99.51 ± 0.95

ac

Intra-day precision (RSD)

0.871

ac

Inter-day precision (RSD)

1.384

d

LOD (μg/mL)

0.08

d

0.24

LOQ (μg/mL)aMean of three determinations. bfor four concentrations of TOBRA (0.6, 0.7, 2.0
and 2.5 μg/mL). cfor three concentrations of TOBRA (1.0, 2.5 and 3.0 μg/mL).
Calculated as follow; LOD = 3.3 × Standard deviation of intercept / slope and
LOQ = 10 × Standard deviation of intercept / slope

El‑Maraghy et al. BMC Chemistry(2024) 18:131

Page 10 of 13

Table 3 Determination of Tobramycin in preparations and the application of standard addition technique by the proposed method
Pharmaceutical preparations

Tobrin®
Ophthalmic solution
BN: 180193*% Found ± SD

100.54 ± 1.783

Claimed taken:
μg/mL

Standard addition technique
Pure added μg/mL

Pure Found μg/
mL

0.45

0.25

0.25

99.54

0.45

0.46

102.221.0

1.00

100.58Mean ± SD

Tobrin®
Ophthalmic ointment
BN: 2010036

98.05 ± 0.262

1.00*% Recovery

100.78 ± 1.35

0.50

0.49

1.0

0.99

99.66

2.0

2.01

100.51

Mean ± SD

98.72

99.63 ± 0.89

*Average of three determinations

Table 4 Determination of tobramycin in spiked human plasma
by the proposed method

Table 5 Statistical comparison between the proposed and thereported methods for determination of Tobramycin

Added (μg/mL)

Parameter

Proposed method

Reportedmethod
[4]a

Mean

99.51

100.74

SD

0.950

0.505

N

4

4

Variance

0.903

0.255

t-test*2.295 (2.447)

F*

3.541(15.439)

Found (μg/mL)*Recovery%

0.50

0.51

102.00%1.0

1.01

101.00%5.0

5.1

102.00%Mean ± SD

101.67 ± 0.577*Average of three determinations

equation was calculated. The method was linear in therange (0.35–4 μg/mL). The accuracy results were satisfactory expressed by the accepted mean recovery(99.51 ± 0.95). The method was precise intra-daily andinter-daily, confirmed by the RSD values above 2 units.
The low values of LOD and LOQ, 0.08 and 0.24 μg/mL
respectively, proved the high sensitivity of the method,
Table 2.
Application of the proposed method for analysisof pharmaceutical preparations and spiked human plasma

The proposed method was applied for the determinationof TOBRA in Tobrin® ophthalmic solution and ointment.
The obtained recovery of TOBRA, (Table 3), revealed theabsence of excipients interference. The selectivity of theprocedure was evaluated by applying the standard addition technique. The ophthalmic route of TOBRA leads
to systemic absorption that may cause some side effects
such as headache and laryngospasm [53], so the drug
plasma concentration needs monitoring. Consequently,
the developed method was applied by spiking TOBRA tohuman plasma samples at three levels, Table 4.
Statistical comparison

The statistical comparison of the results obtained by
the proposed and colorimetric reported method [4] forthe determination of TOBRA is shown in (Table 5). The*

Figures in parenthesis are the corresponding theoretical t and F values at(p = 0.05)

a

TOBRA reacts with diphenylamine to form a blue colored complex whose color
intensity is measured at 635 nm

results showed that there was no significant difference
between the two methods in accuracy and precision
terms as the calculated t and F values were less than thetheoretical ones.
Green assessment of the developed and reported methods

The green character of the developed method wasassessed and compared to colorimetric reported methods
using Green Analytical Procedure Index (GAPI) and theAnalytical GREEnness calculator (AGREE) tools. GAPI
tool classifies the greenness of each stage of the analytical method using a scale of three colors representing thedegree of environmental impact [28, 54]. For our developed method, the green color dominates. There are four
yellow segments (1, 9, 10 and 11) and four red segments(5, 6, 7 and 14). The green character of the two reported
methods for the determination of TOBRA using SDSAgNPs [23] and using diphenylamine [4] was comparedto the developed one. The Reported method [23] has six
red segments due to the extraction step of milk samples,utilization of a large volume of solvents and consequently

El‑Maraghy et al. BMC Chemistry(2024) 18:131

Page 11 of 13

Table 6 Greenness assessment of the proposed and reported methods for determination Tobramycin by GAPI and AGREE tools
Category

Proposed method

Reported method [23]

Reported method [4]Sample collection [1]At-lineAt-lineAt-lineSample preservation [2]

None

None

None

Sample transport [3]

None

None

None

Sample storage [4]

None

None

None

Type of method: direct or indirect [5] Indirect

Indirect

Indirect

Scale of extraction [6]Macro-extraction

Macro-extraction

Macro-extraction

Solvents/reagents used [7]Non-green solvents

Non-green solvents

Non-green solvents

Additional treatments [8]

None

None

Derivatization required

Amount [9]10–100 mL> 100 mL

> 100 mL

Health hazard [10]Acetic acid: NFPA health hazard = 3
Chloroform: NFPA health hazard = 2
AgNps: NFPA health hazard = 0

SDS: NFPA health hazard = 2
Chloroform: NFPA health hazard = 2

Acetic acid: NFPA health hazard = 3
Diphenylamine: NFPA healthhazard = 2
Hydrochloric acid: NFPA healthhazard = 3

Safety hazard [11]Acetic acid: flammability = 2
Chloroform: flammability = 0
AgNps: flammability = 0

SDS flammability = 3
Chloroform: flammability = 0

Acetic acid: flammability = 2
Diphenylamine: flammability = 1
Hydrochloric acid: flammability = 0

Energy [12]≤ 0.1 kWh/ sample≤ 0.1 kWh/ sample≤ 0.1 kWh/ sample

Occupational hazard [13]Hermetic sealing of analytical process

Waste [14]> 10mL> 10mL> 10mL

Waste treatment (15)Recycling

None

None

Quantification

Yes

Yes

Yes

GAPI

AGREE

producing a large amount of waste, which was nottreated. Reported method [4] has seven red segments dueto the same causes as the first reported method in addition to using the derivatization step, which is not considered green treatment. The assessment results of GAPI
are listed in Table 6 showing that the developed method
is greener than the two reported methods. The newly
launched Analytical GREEnness calculator (AGREE)
allows automatic, rapid, and informative assessment
depending on the 12 principles of green analytical chemistry (SIGNIFICANCE) [29]. According to AGREE pictograms, shown in Table 6, the final score of the developed
method is higher than the other two reported methods

with a value of 0.74, which is mainly due to the larger
amount of solvent used and the presence of the derivatization step in reported method [4] which is not considered a green step. In conclusion, the proposed method
has a higher green profile than the reported methods. A
graphical representation of the greenness study is shownin (Supplementary material Fig. S7).

Conclusion
In this work, an eco-friendly, simple, rapid, and sensitive optical sensor was developed and validated forthe determination of non-UV-absorbing drug TOBRAEl‑Maraghy et al. BMC Chemistry(2024) 18:131

using PVP-AgNPs. The optimization of the reaction
conditions to get maximum absorbance quenching wasdone using a factorial design by studying three factors: PVP-AgNPs concentration, pH, and buffer type.
PVP-AgNPs concentration of (215.74 μg/mL) and theuse of Britton-Robinson buffer of pH 9 gave the mostsignificant effect. The proposed method was linear in
the range of (0.35–4 μg/mL). The method is sensitive
with LOD = 0.08 μg/mL and permits rapid analysis of
TOBRA without the need for derivatization nor sample pre-treatment. The method is greener compared to
the reported colorimetric methods according to GAPI
and AGREE tools. The interaction between the drug
and PVP-AgNPs was assessed by the molecular docking
software. Finally, the optical sensor allows the determination of TOBRA in different pharmaceutical dosage
forms (eye solution and ointment) and spiked humanplasma with good recovery. Being green, simple, nonexpensive, rapid, and utilizing the spectrophotometer,
which is available in every lab, it could be used for thequality control tests of TOBRA in bulk powder andpharmaceutical formulations.
Abbreviations
AGREE	Analytical GREEnness calculator
Ag-NPs	Silver nanoparticles
ATR​	Attenuated total reflectance
EBC	Exhaled breath condensate
GAPI	Green Analytical Procedure Index
LC	Liquid chromatography
PVP	Polyvinyl pyrrolidone
SDS	Sodium dodecyl sulfate
SPR	Surface Plasmon resonance
TEM	Transmission electron microscopy
TOBRA	Tobramycin

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​024-​01234-y.
Spplementary Material 1.
Acknowledgements
The authors gratefully thank Modern Science and Arts University for providing
infrastructure and instrumental facility to carry out this work.
Author contributions
C.E. Supervision, methodology, writing, review and editing. P.M. Formal
analysis, methodology, conceptualization, investigation, visualization, andwriting-original draft. R.H. methodology, results investigation and interpreta‑tion. M.A. Supervision, methodology, review and editing and N.F. Supervision,
methodology, review and editing. All authors revised and approved the final
manuscript.
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB). The authors did not receive support from any organization for thesubmitted work.

Page 12 of 13

Availability of data and materials
All data generated or analyzed during this study are included in this publishedarticle.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Received: 8 October 2023 Accepted: 25 June 2024

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