(2024) 18:115
Elgendy et al. BMC Chemistry
https://doi.org/10.1186/s13065-024-01193-4

BMC Chemistry

RESEARCH

Open Access

Determination of levofloxacin, norfloxacin,
and moxifloxacin in pharmaceutical dosage
form or individually using derivative UV
spectrophotometry
K h. Elgendy1*, M. Zaky1, Alaa Eldin mohamed Mahmoud altorky1 and S. Fadel1   

Abstract
Purpose In this study, first, second, third, and fourth-order derivative spectrophotometric methods utilizing
the peak—zero (P—O) and peak-peak (P—P) techniques of measurement were developed for the determinationof levofloxacin, norfloxacin, and moxifloxacin. These methods were applied to their combined pharmaceutical dosage
form or individually for levofloxacin, norfloxacin, and moxifloxacin.
Methods Linearity was established in the concentration range of 2–20 µg/mL. The procedures are simple, quick,and precise. The developed methods are sensitive, accurate, and cost-effective, demonstrating excellent correlation coefficients (R2 = 0.9998) and mean recovery values ranging from 99.20% to 100.08%, indicating a high levelof precision.
Results The developed approach was effectively employed to determine the levofloxacin, norfloxacin, and moxifloxacin content in commercially available pharmaceutical dosages.
Conclusions Statistical analysis and recovery tests confirmed the method’s linearity and accuracy. The results suggest that this method can be utilized for routine analysis in both bulk and commercial formulations. The simplicity,
accuracy, and cost-effectiveness of the developed methods make them valuable for pharmaceutical analysis.
Keywords Levofloxacin, Norfloxacin, Moxifloxacin, Validation, Simultaneous estimation, Derivative Uvspectrophotometry
Introduction
Derived UV-spectrophotometry is a method that provides both qualitative and quantitative information fromspectra in disputable bands. It involves using the first or
higher derivatives of absorbance concerning wavelength
for analysis purposes [1]. Derivative spectroscopy was*Correspondence:
K h. Elgendy
El_gendykh@yahoo.com
1
Chemistry Department, Faculty of Science, Zagazig University, Zagazig,
Egypt

first introduced in the 1950s and demonstrated various
advantages; however, it received limited attention due
to the challenges of generating derivative spectra withearly UV–Visible spectrophotometers. [2] The techniquegained traction with the advent of microcomputers in thelate 1970s. These computers allowed for the quick, easy,
and reproducible generation of derivative spectra using
mathematical methods. This technological advancement
significantly increased the application of the derivativetechnique. IN this application note, we will delve into
the mathematics and generation methods of derivative
spectroscopy in a concise manner. Computer-generated© 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.

Elgendy et al. BMC Chemistry(2024) 18:115

examples will be used to illustrate the features and applications of this technique (see Fig. 1) [20].
Derived UV‑spectrophotometry: application and methods
overview

Derived UV-spectrophotometry, employing first to
higher-order derivatives, provides both qualitative andquantitative information from spectra. The distinguishing feature of second-order derivatives is a negative bandwith a minimum at the same wavelength as the maximum on the zero-order band, along with positive satellite bands. Fourth-order derivatives display a positive
band. Even-order derivatives exhibit a strong negative or
positive band with a minimum or maximum at the same
wavelength as the absorbance band’s maximum. The
number of observed bands is one more than the derivative order [3–5]. Second-order Derivative: Negative bandat the same wavelength as the zero-order maximum, withpositive satellite bands. Fourth-order Derivative: Positive
band. Even-order Derivatives: Strong negative or positive
band with a minimum or maximum at the same wavelength as the absorbance band’s maximum [6].

Methods of derivative spectra generation
Optical and electronic techniques

Wavelength Modulation: Involves rapid modulation ofincident light wavelength over a narrow range. Suitablefor generating first and second derivatives. Dual Wavelength Spectrophotometer: Produces first-derivative
spectra by scanning with each monochromatic wavelength separated by a small constant difference.
Mathematical techniques

Easily calculated and recalculated with different parameters. Smoothing techniques can enhance signal-to-noise
ratio. The determination by the “peak—zero” (P—O) and

Fig. 1 Absorbance and derivative spectra of a Gaussian band

Page 2 of 21“peak—peak” (P–P) techniques involves analyzing thederivative spectra to obtain specific information about
the drugs being studied. Here’s a more detailed explanation for both techniques:
Peak—Zero (P—O) TechniqueIn the "peak—zero" technique, the analysis is based on
measuring the amplitude from the maximum to the zeroline or from the minimum to the zero line of the derivative spectra. The derivative spectra represent the rate ofchange of absorbance with respect to wavelength. Peaks
or valleys in the derivative spectra correspond to specificfeatures in the original absorbance spectrum. The "peak—zero" technique involves measuring the amplitude of aparticular peak or valley in the derivative spectrum fromits highest or lowest point to the zero line. This amplitude can be related to the concentration of the substancebeing analyzed, and the technique is used to determine
the presence and concentration of specific compounds.
Peak—Peak (P‑P) TechniqueIn the "peak—peak" technique, the analysis is based on
measuring the amplitude from one peak to another peakin the derivative spectrum. Similar to the “peak—zero”
technique, the derivative spectrum is examined for peaks
and valleys, and the amplitude is measured between two
specific peaks. This amplitude can be used for quantification and is related to the concentration of the analyzed
substance. The “peak—peak” technique provides an alternative way to extract information from the derivative
spectra, allowing for a comprehensive analysis of the features present.
Both techniques essentially use the characteristics ofthe derivative spectra to quantify the concentration ofsubstances. By measuring the amplitudes in different
ways, analysts can choose the most suitable approachElgendy et al. BMC Chemistry(2024) 18:115

based on the specific features of the derivative spectra
and the compounds being studied. These techniques arepart of the broader field of derivative spectrophotometry, offering advantages in terms of sensitivity and selectivity in the determination of drugs and other chemical
substances.
Fluoroquinolones and antibacterial chemotherapy

Fluoroquinolones, a class of antibiotics structurally similar to nalidixic acid, exhibit enhanced antibacterial activity. Improved potency, broader antibacterial spectrum,and favorable pharmacokinetic properties contribute totheir clinical significance. Widely distributed in the body,
fluoroquinolones are effective against various infections,
including urinary, respiratory, gastrointestinal, skin, soft
tissue, bone, and sexually transmitted infections. Theease of oral therapy adds to their advantages.
Quinolones and fluoroquinolones

Quinolones, structurally similar to nalidixic acid, achieve
bactericidal action by blocking bacterial DNA gyrase.
Early quinolones had limitations, including a narrowspectrum, low potency, high resistance rates, low serum
concentrations, and short half-lives. Fluoroquinolones
overcome these limitations with a broader antibacterial range, increased potency, low resistance rates, highoral bioavailability, significant tissue penetration, andextended elimination half-lives. Potential side effects
include gastrointestinal disturbances, rashes, central
nervous system stimulation, cartilage damage, eye toxicity, teratogenicity, and spermatogenesis impairment[7, 8]. Newer Fluoroquinolones characterized by broadspectrum bactericidal action, excellent oral bioavailability, good tissue penetration, and favorable safety andtolerability. Classified into four generations based on
antibacterial spectrum and therapeutic indications. The
extended antibacterial spectrum of the most recent fluoroquinolones allows for a diverse range of applications.
Spectroscopic Analysis of Levofloxacin, Norfloxacin, andMoxifloxacin, antibiotics used against various bacterial
species, were studied using fluorescence and UV–visible
absorption spectroscopy. Solvent polarity and drug concentration influenced fluorescence quantum yields, lifespan, and non-radiative decay. Fluorescence quenching
techniques revealed binding mechanisms with caffeine,indicating ground state complexes and contributions
from electrostatics, hydrogen, and Van der Waals forces[9–12].
Recently, UV–Visible spectroscopy and density functional theory (DFT) techniques, our investigation delved
into the development of charge-transfer (CT) complexes.
Our focus centered on exploring the interaction of iodinewith aniline and its derivatives in CCl4 across varying

Page 3 of 21

temperatures (293.15–308.15 K). The calculation of formation constants (KCT) using the Benesi-Hildebrandplot revealed the influence of temperature changes anddonor molecule structure on KCT values. Both experimental and DFT UV spectra uncovered an additional CT
band, with the elongation of I2 acceptor bonds facilitating the observation of the interaction between I2 and aniline derivatives [13]. Introduced an innovative titrimetricapproach (method A) for the precise determination of
Ciprofloxacin Hydrochloride (CIP-HCl). This approach
involved the in situ bromination of CIP-HCl through theinteraction of acid with the bromate-bromide combination. Additionally, a UV-Spectroscopic analysis (methodA), as per previous literature, demonstrated adherence
to Beer’s law within the concentration range of 3.5–
11.5 g ­mL−1. Titrimetric analysis extended the determination range to 5.0–70.0 mg CIP-HCl. Comparative analysisof accuracy and precision with the reference method(method B) showcased negligible differences in results.
Method A successfully determined three distinct dosage forms of CIP-HCl [14]. A novel, rapid, specific, andeconomical UV spectrophotometric method was established for the quantification of levofloxacin in both pureform and pharmaceutical formulations. This techniqueemploys a distinct solvent mixture (water: methanol: acetonitrile) and measures absorbance at a pre-determinedwavelength (292 nm). It was demonstrated to be linear across a broad range of levofloxacin concentrations(1.0–12.0 mg/mL), exhibiting a commendable correlation
coefficient ­(R2 = 0.9998) and exceptional mean recovery(99.00–100.07%). [15, 16]. Study investigated the application of highly crystalline ­TiO2 nanoparticles in thephotocatalytic degradation of levofloxacin. Synthesized
through a sol–gel method, the ­TiO2 nanoparticles underwent comprehensive assessment for morphological,
structural, compositional, thermal, and optical characteristics. The in-depth research highlighted the nanoparticles’ high density, exceptional crystallinity, and favorable
optical characteristics, contributing to efficient photocatalytic degradation [17]. Employing a straightforwardhydrothermal and sol–gel synthesis, we created carbon
dots (C-dots), titanium dioxide ­(TiO2) quantum dots,and ­TiO2/C-dots. Thorough characterization encompassing crystallinity, structure, morphology, thermal stability, and optical properties confirmed the great quality ofthe produced photo catalysts. The nanocomposites displayed an average size of 12 nm, with C-dots uniformly
dispersed across T­ iO2 quantum dots [18]. Addressing
contemporary environmental challenges, our emphasis
centered on the need for photocatalytic materials withsuitable structural and morphological architectures. The
creation of Z-scheme hetero junctions emerged as an
ideal strategy to overcome the limitations associated with

Elgendy et al. BMC Chemistry(2024) 18:115

single-component or conventional heterogeneous catalysts. Our discussion highlighted the diverse applicationsof these materials, particularly in the effective removal oforganic compounds from wastewater [19]. Presenting asimple, quick, and cost-effective technique, we detailed
the preparation of MIL-100(Fe) using a sealed autoclave
and a solid-state reaction. Extensive characterization
using PXRD, FTIR, SEM, EDX, TGA, BET surface area,and zeta potential analysis provided insights into theproperties of the as-prepared MIL-100(Fe). The resulting
MIL-100(Fe) effectively served as an adsorbent for the
extraction of the antibiotic levofloxacin from the aqueous phase [20]. More research technique were reviewed[21–37].

Materials and methods
Devices

UV–Visible double-beam spectrophotometric, Shimadzu
1800 Origin, Kyoto, Japan, was employed. Absorbancemeasurements were conducted using Uv. Probe 2.34,
Model Shimadzu Origin Kyoto Japan, with matched
quartz cells. Additional equipment included a laboratory oven-sonorous, a sensitive electronic balance, anultrasonic device (ultrasonicator)-sonorex, a water bath
(Karl K olb), and a hot plate with magnetic stirrer (LMS1003, Daihan Lab Tech) from Germany. Precision measurements were facilitated by micro pipettes (1–10 µl and100 µl) originating from Switzerland. Computational
aspects were handled on a Dell computer from Germany
and China.
Chemical materials

Levofloxacin, Norfloxacin, and Moxifloxacin were generously supplied by Arti (India). Eipico Company, locatedPage 4 of 21

in the 10th of Ramadan City, Egypt, provided pharmaceutical-grade excipients, including Citric acid, Magnesium stearate, Vinyl pyrrolidone, Starch, Talc, andLactose. Deionized water utilized in the analysis underwent preparation through reverse osmosis and filtration
via a 0.45 μm Millipore filter (Millipore Company, USA).
Market sample

Tablet forms of Levofloxacin, Norfloxacin, and Moxifloxacin were randomly selected from the market. The
recently developed and approved method was applied
to analyze sample solutions against the reference standard. Quantities of Levofloxacin, Norfloxacin, and Moxifloxacin were determined using data from the marketed
products.
Levoxin (250 mg Levofloxacin) by ALAR LABORATORIES PVT LIMITED, Composition: Levofloxacin: 250 mg, and excipients: maize starch, Magnesium
stearate, Polyethylene glycol, Povidone K30, Yellow Tartrazine, and Talc.
Epinor (400 mg Norfloxacin) by EIPICO. Composition:
Norfloxacin: 400 mg Excipients: maize starch, Magnesium stearate, Polyethylene glycol, Povidone K90, Titanium dioxide, and Talc.
Delmoxa (400 mg Moxifloxacin) by DELTA PHARMA.
Composition: Moxifloxacin: 400 mg, and excipients:maize starch, Magnesium stearate, Lactose, Povidone
K90, Talc, and Iron oxide red.
Solutions

Solution (1000 µg/mL): Prepared by dissolving 0.1 g ofeach substance in 100 ml of 0.1 mol/L HCl solution. Solution (100 µg/mL): Obtained by diluting the stock solution
in methanol and further dilution in a volumetric flask to

Fig. 2 Spectrums of levofloxacin, norfloxacin, and moxifloxacin in zero orderElgendy et al. BMC Chemistry(2024) 18:115

Page 5 of 21

Table 1 Short-term Stability
Drugs

Concentration
declared (mg/mL)

Concentrationfound(mean ± SD)

RSD (%)

Averagepotency(%)

Levofloxacin

21.96 ± 0.05

0.295

98.19

4

3.95 ± 0.05

0.146

98.80

6

5.97 ± 0.05

0.097

99.41

2

1.94 ± 0.05

0.295

98.20

4

3.90 ± 0.05

0.265

99.90

6

5.93 ± 0.05

0.098

100.052

1.95 ± 0.05

0.295

98.19

4

3.96 ± 0.05

0.150

99.50

6

5.98 ± 0.05

0.255

98.90

Norfloxacin

Moxifloxacin

Table 2 Recovery/accuracy for three different concentrations of
Levofloxacin, Norfloxacin, and Moxifloxacin
Dosage form

Label claim

levofloxacin

0.250 mg

Norfloxacin

Moxifloxacin

Amountadded (%)

Recovery (%)

80

99.80

100

99.45

120

99.60

80

100.050.400 mg

100

99.70

120

100.010.400 mg

80

99.5

100

100.08120

99.20

Stability study
Solvent selection and standardization

In the preliminary trial, five different solvent compositions were considered, including distilled water, methanol, methanol: distilled water (1:1), 0.1 N HCl, andphosphate buffer (pH 7.1). After evaluation, methanol:
distilled water (1:1) was chosen as the suitable medium
due to its ease of sample preparation, the drug’s solubility, and cost-effectiveness. The wavelength maximum forlevofloxacin, norfloxacin, and moxifloxacin was identified
at 287 nm,291 nm, and 294 respectively A stock solutionof the levofloxacin, norfloxacin, and moxifloxacin standard were prepared with an approximate concentration of
5 μg/mL and subjected to sonication for 4 min in a sonicator bath.
Linearity

The response function was determined by preparing
standard solutions at ten different concentration levels ranging from 2 to 20 μg/mL using UV/Vis derivative
spectrophotometric methods. The limit of detection(LOD) and limit of quantitation (LOQ) were calculated
based on the standard deviation of the Y-intercept andthe slope of the calibration curve following the International Council for Harmonisation (ICH) guidelines [20].
The formulas used were LOD = 3.3 × (standard deviation of Y-intercept/slope of the curve) and LOQ = 10 × 2(standard deviation of Y-intercept/slope of the curve).
Accuracy

achieve a concentration of 100 µg/mL in 0.1 mol/L HCl
solution (2–20 µg/mL). Additives (1000 µg/mL): Solution
containing Levofloxacin, Norfloxacin, Moxifloxacin, andother additives, prepared in 100 mL of 0.1 mol/L HCl.

A study was conducted using pre-formulated granules
containing pure Levofloxacin, Norfloxacin, and Moxifloxacin, along with common excipients. The calculation
was based on the label claim and the average weight of
the final product. A previously established dilution pattern was followed for the granules, resulting in three
concentrations 80%, 100%, and 120% of the reference
solution. Three replicate samples were prepared at each

Table 3 Precision Study levofloxacin
Sample No% Assay

Intra-day assay

Inter-day assay(repeatability)

1st h

3rd h

8th h

1st day

3rd day

5th day

7th day

1

100.8

99.25

100.2599.85

101.52100.5899.52

100.252

101.8599.65

100.3599.65

98100.52

100.2

100.6

3

101.6599.4

100.45

100.2

99.5

99

99.8

100.2

4

101.6

99.5

100.3

99.68

98.8

102.5

102.5

99.5

5

101.5599.58

100.5899.5

100.25

101.5

99.8

99.6

6

101.4

99.65

100.45

99.85

101.8

99.85

101.5

99.54

Mean ± SD

101.74 ± 0.36

99.54 ± 0.16

100.4 ± 0.085

99.76 ± 0.25

99.87 ± 1.50

100.55 ± 1.43

100.0 ± 1.13

99.9 ± 0.461% RSD

0.361593

0.168325

0.085391

0.252521

1.506906

1.43373638

1.358958

0.460751

Elgendy et al. BMC Chemistry(2024) 18:115

Page 6 of 21

Table 4 Precision Study norfloxacin
Sample No% Assay

Intra-day assay(repeatability)

1st hInter-day assay

3rd h

8th h

1st day

3rd day

5th day

7th day1

100.0799.58

100.25

99.5

100.2

100.2

98.2

100.52

2

100.5

99.5

100.39

99.5

99.8

99.8

100.5

100.2

3

101.5

100.25101.25

101.2

99.2

99.6

100.9

100.254

100.25101.02

100.2

99.5

100.8

101.5

100.8

98.9

5

100.25

99.6

99.85

99.35

101.25

101.8

99.9

99.6

6

100.2

99.58

99.8

99.7

100.18

100.2

100.9

99.2

Mean ± SD

100.25 ± 0.52

99.59 ± 0.60

100.2 ± 0.52

99.5 ± 0.69

100.19 ± 0.72

100.2 ± 9.13

100.65 ± 1.05

99.9 ± 0.646% RSD

0.527462

0.604795

0.524404

0.698868

0.723061

0.91305349

1.050714

0.646233

Table 5 Precision Study moxifloxacin
Sample No% Assay

Intra-day assay

Inter-day assayrd(repeatability)

1st h

3 h

8th h

1st day

3rd day

5th day

7th day

1

99.5

99.5

100.2

99.5

100.6

101.25

99.5

99.5

2

100.2

99.8

100.25

99.25

99.2

99.2

99.7

99.2

3

100.6

100.2

100.5

100.6

100.5

99.4

101.25

99.6

4

101.5

100.8

101.6

99.7

100.6

99.8

100.4

100.52

5

100.3

101.6

99.75

99.2

100.7

100.5

99.25

101.2

6

99.25

99.8

99.65

99.9

101.25101.25

98.9

100.6

Mean ± SD

100.25 ± 0.80

100.0 ± 0.78

100.2 ± 0.70

99.6 ± 0.51

100.6 ± 0.67

100.15 ± 0.90

99.6 ± 0.85

100.06 ± 0.78

% RSD

0.834666

0.561991

0.654949

0.587899

0.684957

0.92590046

0.792017

0.5692978

concentration level, and the percent recovery at eachlevel (n = 10) was determined.
Precision

By repeatedly scanning levofloxacin, norfloxacin, andmoxifloxacin standard samples (n = 6) without altering
the parameters of the suggested derivative spectrophotometric method, the instrumental precision, expressed
as relative standard deviation (RSD), was verified. Dataon intra-assay and inter-assay precision (RSD) were collected in the lab on days 1, 3, and 7 as well as on several
days over the course of a week (days 1, 3, 5, and 7). The
standard deviation of the measurements divided by themean of the measurements multiplied by 100 is how the
RSD percentage is computed.
Assay

Three marketed brands (Levoxin, Epinor and Delmoxa) were tested using this method, with levofloxacin at 287 nm, norfloxacin at 281 nm and moxifloxacin
at 294 nm, using the following formula: active ingredient content (mg/ tablet). = (sample absorbance/standard
absorbance) × (standard weight/sample weight) × average
weight × standard intensity/100.

Samples generated for repeatability research werestored at room temperature for 24 h before undergoing
testing for short-term stability the following day.
Specificity in the presence of excipients

An analytical method’s specificity is intended to quantitatively identify the analyte as a constituent that is
anticipated to exist in the sample matrix. Popular additives, such as lactose, povidone K30, magnesium stearate,
and purified talc, are combined in the right proportions
per test protocol and dissolved in a solvent system thatmatches the additive weight during sample preparation.
First, the nominal concentration of the drug is combined with various additive concentrations (80–120%of the nominal concentration in the test formulation).
After increasing the additive’s nominal concentration
to different drug concentration levels (80–120% of thetest formulation’s nominal concentration), absorption isassessed. To calculate how much medication is needed,
perform some calculations. The response of commercially available products, standard of levofloxacin, norfloxacin, and moxifloxacin, and additives under stressconditions was compared with the response of the same
solutions under stress conditions in order to determine

9.75

10

10

LactoseVinyl pyrrolidone

9.95

9.9

9.8

10

StarchMagnesium stearate 10

99

99.5

97.5

98

99

98.5

10

10

10

10

10

9.77

9.86

9.97

9.92

9.95

9.85

10

9.9

9.85

10

10

Taken (µg/mL) Found (µg/mL)

Taken (µg/mL) Found (µg/mL)

Talc

λ(287)nm

Rec % Second D2

λ(287)nmFirst D1

Citric acid

Additives

98.5

97.7

98.6

99.7

99.2

99.5

10

10

10

10

10

10

9.87

9.9

9.98

9.85

9.9

9.85

Taken (µg/mL) Found (µg/mL)

λ(287)nm

Rec % Third D3

Table 6 The suggested method’s effect of additives (100 µg/mL) on the drug levofloxacin determination

98.5

98.7

99

99.8

98.5

99

10

10

10

10

10

10

9.8

9.95

9.9

9.85

9.77

9.85

Taken (µg/mL) Found (µg/mL)

λ(287)nm

Rec % Fourth D4

98

99.5

99

98.5

97.7

98.5

Rec %

Elgendy et al. BMC Chemistry(2024) 18:115
Page 7 of 21

9.85

10

10

LactoseVinyl pyrrolidone

9.9

9.8

9.85

10

StarchMagnesium stearate 10

98

99

98.5

98.5

98

99

10

10

10

10

10

9.95

9.85

9.8

9.9

9.90

9.95

10

9.80

9.9

10

10

Taken (µg/mL) Found (µg/mL)

Taken (µg/mL) Found (µg/mL)

Talc

λ(287)nm

Rec % Second D2

λ(287)nmFirst D1

Citric acid

Additives

99.5

99.5

98.5

98

99.0

99.0

10

10

10

10

10

10

9.85

9.85

9.95

9.90

9.85

9.90

Taken (µg/mL) Found (µg/mL)

λ(287)nm

Rec % Third D3

Table 7 The suggested method’s effect of additives (100 µg/mL) on the drug norfloxacin determination

99.0

98.5

98.5

99.5

99

98.5

10

10

10

10

10

10

9.7

9.9

9.95

9.98

9.75

9.75

Taken (µg/mL) Found (µg/mL)

λ(287)nm

Rec % Fourth D4

97

99

99.5

99.8

97.5

97.5

Rec %

Elgendy et al. BMC Chemistry(2024) 18:115
Page 8 of 21

9.95

10

10

LactoseVinyl pyrrolidone

9.95

9.85

9.75

10

StarchMagnesium stearate 10

99.5

99.5

99.5

97.5

98.5

99

10

10

10

10

10

9.77

9.78

9.92

9.82

9.8

98.5

10

9.95

9.9

10

10

Taken (µg/mL) Found (µg/mL)

Taken (µg/mL) Found (µg/mL)

Talc

λ(287)nm

Rec % Second D2

λ(287)nmFirst D1

Citric acid

Additives

98.5

97.7

97.8

99.2

98.2

98.0

10

10

10

10

10

10

9.87

9.78

9.88

9.95

9.8

9.75

Taken (µg/mL) Found (µg/mL)

λ(287)nm

Rec % Third D3

Table 8 The suggested method’s effect of additives (100 µg/ml) on the drug moxifloxacin determination

97.5

98.7

97.8

98.8

99.5

98

10

10

10

10

10

10

9.8

9.8

9.95

9.95

9.97

9.88

Taken (µg/mL) Found (µg/mL)

λ(287)nm

Rec % Fourth D4

98

98.0

99.5

99.5

99.7

98.8

Rec %

Elgendy et al. BMC Chemistry(2024) 18:115
Page 9 of 21

Elgendy et al. BMC Chemistry(2024) 18:115

Page 10 of 21

Table 9 Forced degradation study of levofloxacin, norfloxacin, and moxifloxacin
Levofloxacin

Hydrolysis

Photolytic
Oxidation
Thermal (solid state)*

Norfloxacin% Assay ofStandard% Assay of
product

Moxifloxacin% Assay ofstandard% Assay of
product% Assay ofstandard% Assay of
product

Neutral hydrolysis at RT*

7th

90.25

103.4589.98

104.5499.5

103.54

Neutral hydrolysis at 60°

7th

125.93100.5

135.93101.78105.93101.78Acid hydrolysis at RT*

7th

89.69

98.5

89.69

94.04

99.69

94.04

Acid hydrolysis at 60°

5th

99.59

85.58

93.59

83.37

96.59

83.37

6th

112.93142.35112.93142.35112.93142.35

Base hydrolysis at RT*

3rd

60.79

60.5

59.79

66.78

60.5

66.78

Base hydrolysis at 60°

1st

27.8

82.5

37.8

72.78

55.05

72.78

Dark (solution state)

14th

118.64105.68108.64106.68103.6

106.68Light (solution state)

14th

103.48122.28102.48142.28102.48132.2830% ­H2O2 at RT*

7th

85.54

102.7280.54

100.72

90.54

100.7230% ­H2O2 at 60°

7th

126.76109.29106.76108.29102.76108.29

RT*

14th

106.2

99.18

102.2

99.08

102.2

99.5

60°

14th

101.72104.83112.40104.83111.72101.83

RT Room Temperature

the stability-indicating properties of the developed UV/
visible method. This analysis is a component of a study
on forced degradation.
Forced degradation study

The drug material and one of the three commerciallyavailable drug products were both forced to degrade
under various stress conditions in this study. These conditions included neutral, acidic, and base hydrolysis as
well as oxidative, photolytic, and thermolytic stress. Specifically, the drug substance and drug product degradation was carried out in the solid state. The drug substanceor drug product is dissolved in distilled water, aqueous
HCl/NaOH/H2O2, or a solvent until a concentrationof 50 μg/mL is reached to be able to start a degradation
study. Subsequently, these solutions were diluted using amethanol: distilled water (1:1) solvent to a concentrationof roughly 5 μg/mL. The degradation was then observedby performing a UV/visible spectrophotometric, a spectral scan in the 220–350 nm range, and measuring theabsorbance on various days. Hydrolysis tests were performed to evaluate the impact of different conditions on
the drug substance and drug product. For duration ofseven days, neutral hydrolysis using distilled water was
conducted at room temperature and 60 ℃. Using 0.1 N
HCl, acid hydrolysis was carried out for seven days at
60 ℃ and room temperature. In contrast, base hydrolysis used 0.1 N NaOH solutions at 60 ℃ and room temperature, but only for two days. Sample solutions of
the drug substance and drug product were exposed to3% ­H2O2 at room temperature and 60 ℃ for a week inorder to assess oxidative stress. Over 14 days, photolytic
stress experiments were conducted, and both light anddark conditions were observed. The drug substance anddrug product were subjected to controlled oven temperatures—room temperature and 70 ℃, specifically—for 14 days to induce thermal stress. It is significant toremember that all samples under stress were given placebo preparations.
Determination of levofloxacin, norfloxacin,
and moxifloxacin in pharmaceutical dosage

Weighed and milled were ten tablets of Levonex (0.250 gof levofloxacin), Epinor (0.400 g of norfloxacin), andDeltamox (0.400 g of moxifloxacin). Each tested drug’s
50 mg of homogenized powder was carefully weighed
and added to a 50 mL volumetric flask along with 20 mL
of 0.1 mol/L HCl. 0.1 mol/L HCl was used to dilute thevolumes to 50 ml after the combinations were shaken
for 20 min. The resulting solutions were then filtered.
Then, using 0.1 mol/L HCl, 1 mL of each clear solution
was diluted to 10 mL. These solutions’ volumes—0.5 mL,
0.5 mL, and 1.0 mL for the levofloxacin, norfloxacin,
and moxifloxacin solutions, respectively—were put into
10 mL volumetric flasks and diluted with 0.1 mol/l HCl
until the proper volume was reached. For these solutions, derivative spectra at the first, second, third, andfourth orders were noted. The concentrations of Levofloxacin, Norfloxacin, and Moxifloxacin in the studied
sample solutions were determined by interpolating theElgendy et al. BMC Chemistry(2024) 18:115

Page 11 of 21

Fig. 3 First (l) second (2), third (3) and fourth (4) derivative spectra of levofloxacin

corresponding calibration curves, and the amplitudes ofthe minimum and maximum were graphically measured.

Results
Method development and optimization

Levofloxacin, Norfloxacin, and Moxifloxacin demonstrated high solubility in 0.1 mol HCl.The optimized
solvent composition was water (5): methanol (5) for
favorable UV analysis. The absorption spectra were
scanned in the range of 220–350 nm, revealing maximum absorption wavelengths (λ max) for Levofloxacin
at 287 nm, Norfloxacin at 291 nm, and Moxifloxacin at
294 nm.

Method validation
Linearity and range

The calibration curve exhibited a linear correlation coefficient (R2) exceeding 0.99. Calibration curves for derivative spectra of each drug were constructed, showing
significant clarity in the graphs.
Plotting the graphically measured (mm) amplitudesof the first-, second-, third-, and fourth-order derivative
spectra against the corresponding concentrations of thedrugs under investigation allowed for the constructionof calibration curves. The first, second, third, and fourthorder derivative spectra of the levofloxacin standard solution in 0.1 mol HCI are displayed in Figure 3. They wererecorded at concentrations of 2.0 to 20.0 µg mL of levofloxacin, within the wavelength range of 220–350 nm.

Elgendy et al. BMC Chemistry(2024) 18:115

Page 12 of 21

Fig. 4 First (l) second (2), third (3) and fourth (4) derivative spectra of norfloxacin

The first, second, third, and fourth-order derivative spectra of the norfloxacin standard solution in 0.1 mol HCI
are displayed in Figure 4. The spectra were recorded atconcentrations of 2.0 to 20.0 µg mL of norfloxacin, within
the wavelength range of 220–350 nm. The first, second,
third, and fourth-order derivative spectra of the moxifloxacin standard solution in 0.1 mol HCI are displayedin Figure 5. The spectra were recorded at concentrationsof 2.0 to 20.0 µg mL of moxifloxacin, within the wavelength range of 220–350 nm. Tables l0, 12, and 14 present
the linear equations derived from the regression analysis of the ciprofloxacin hydrochloride, norfloxacin, andofloxacin data, respectively. The data on the determination of the studied fluoroquinolones in tablets, along witha statistical analysis of the outcomes, are displayed inTables 11, 13, and 15.
Limit of detection and limit of quantification

LOD and LOQ values, calculated from linearity studies,indicated high sensitivity of the proposed method in therange of 2-20 µg/mL for Levofloxacin, Norfloxacin, andMoxifloxacin. Are displayed in Figure 2

Elgendy et al. BMC Chemistry(2024) 18:115

Page 13 of 21

Fig. 5 First (l) second (2), third (3) and fourth (4) derivative spectra of moxifloxacin

Stability

Accuracy/recovery

Stability study results (Table 1) indicated that the samples
were stable for 24 h (short-term) at various concentrations, affirming the robustness of the proposed method.
For all three medications at various concentrations (2,4, and 6 mg/mL), the values of the concentration foundgenerally agree with the claimed concentration. All drug
potencies vary from 98.19% to 100.05% on average, whichshows that there is good agreement between the declared
and actual concentrations. The measurements are consistent and show little fluctuation around the mean when
the RSD values are low.

Results within the range of 99.20–100.08% ensure an
accurate method as shown in (Table 2).
Precision

The repeatability and intra-day assay precision (RSD)
was < 1% and the inter-day assay precision was < 2%revealed the proposed method for analyzing Levofloxacin
is both precise and accurate (Table 3).
The repeatability and intra-day assay precision (RSD)
was < 1% and the inter-day assay precision was < 2%revealed the proposed method for analyzing norfloxacin
n is both precise and accurate (Table 4).

Elgendy et al. BMC Chemistry(2024) 18:115

Page 14 of 21

Table 10 Statistical evaluation of the developed method for levofloxacin (standard solution, n = 10)
Derivative

λ (nm)

TechniqueRegression equation

D1

275

Absorbance (p-o)

y = 0.0029x + 0.0095

0.946

D1

297

Absorbance (p-o)

y = − 0.0033x–0.0112

0.934

D1

286–322

Peak(P-P)

y = − 0.0504x–0.1996

0.989

D1

257–286

Peak(P-P)

y = 0.0335x + 0.2049

0.982

D1

257–322

Peak(P-P)

y = − 0.0164x + 0.0024

0.988

D2

305

Absorbance (p-o)

y = 0.0003x + 0.0009

0.969

D2

287

Absorbance (p-o)

y = − 0.0005x–0.0022

0.970

D2

296

Absorbance (p-o)

y = 0.0004x + 0.001

0.988

D2

262–276

Peak(P-P)

y = 0.0023x + 0.0179

0.986

D2

276–297

Peak(P-P)

y = − 0.0066x–0.0117

0.976

D2

297–318

Peak(P-P)

y = 0.0041x + 0.0113

0.987

D2

262–318

Peak(P-P)

y = 0.0003x + 0.0116

0.980

D3

278

Absorbance (p-o)

y = − 0.00006x–0.00021

0.974

D3

296

Absorbance (p-o)

y = 0.00005x + 0.0003

0.985

D3

311

Absorbance (p-o)

y = − 0.00003x–0.00008

0.980

D3

267–287

Peak(P-P)

y = − 0.0007x–0.0043

0.983

D3

287–306

Peak(P-P)

y = 0.0006x + 0.005

0.979

D3

306–317

Peak(P-P)

y = − 0.0001x–0.0003

0.997

D3

267–317

Peak(P-P)

y = 0.0002x–0.0078

0.977

D4

270

Absorbance (p-o)

y = − 0.00001x–0.00008

0.986

D4

287

Absorbance (p-o)

y = 0.00001x + 0.00012

0.993

D4

304

Absorbance (p-o)

y = − 0.00001x–0.00007

0.988

D4

316

Absorbance (p-o)

D4

267–275

Peak(P-P)

y = − 0.00002x–0.00018

0.968

D4

275–296

Peak(P-P)

y = 0.0002x + 0.0006

0.980

D4

296–314

Peak(P-P)

y = − 0.00009x–0.00053

0.991

D4

314–319

Peak(P-P)

y = 0.00001x + 0.00004

0.980

D4

267- 319

Peak(P-P)

y = 0.00006x + 0.00029

0.973

y = 0.000005x–0.000004

Correlation
coefficient

0.993

p-o refers to point-to-origin, P-P refers to peak-to-peak

The repeatability and intra-day assay precision (RSD)
was < 1% and the inter-day assay precision was < 2%revealed the proposed method for analyzing moxifloxacin in n is both precise and accurate (Table 5).
The ability of a method to measure the analyticalresponse accurately in the presence of all possible components is referred to as specificity. Furthermore, confirming the suggested method’s specificity is the sample’sstress analysis. The study aimed to determine the critical variables that will affect the drug product’s stability
and validate the developed method’s ability to indicate
stability. To assess the interference of degradation products in the quantitation of levofloxacin, norfloxacin, andmoxifloxacin, the specificity was determined by ICH
guidelines by subjecting a sample solution and solid
to accelerated degradation by acidic/alkaline/neutralhydrolytic, oxidative, photolytic, and thermal stress conditions. The reported method provides data on specificity for their estimation in the presence of degradants andformulation excipients.
Specificity in the presence of excipients

The specificity of the method was confirmed by comparing spectra of placebo granules and degradation productswith that of accurate samples.
Study effect of additives

First derivative of peak absorbance measurement at wavelength (287) nm, second derivative of peak absorbancemeasurement at wavelength (287) nm, third derivative of
peak absorbance measurement at wavelength (287) nm,
and fourth derivative of peak absorbance measurement at

Elgendy et al. BMC Chemistry(2024) 18:115

Page 15 of 21

Table 11 Statistical analysis of the determination of levofloxacin in levoxin tablet (n = 10)
Derivative

λ (nm)

TechniqueContent of
Levofloxacin (g)Standard Deviation

Variance

Correlation
Coefficient

ConfidenceInterval(RSD)—95%

D1

275

Absorbance (p-o)

0.2538.25*10^-3

9.36*10^-50.81

0.243–0.264

D1

297

Absorbance (p-o)

0.251

8.65*10^-3

7.59*10^-50.34

0.243–0.262

D1

286–322

Peak(P-P)

0.253

7.59*10^-3

7.22*10^-50.15

0.245–0.264

D1

257–286

Peak(P-P)

0.252

8.32*10^-3

8.24*10^-50.24

0.244–0.261

D1

257–322

Peak(P-P)

0.255

8.95*10^-3

7.35*10^-50.75

0.242–0.260

D2

305

Absorbance (p-o)

0.2538.22*10^-3

8.23*10^-50.640.243–0.259

D2

287

Absorbance (p-o)

0.2528.45*10^-3

8.25*10^-50.33

0.244–0.262

D2

296

Absorbance (p-o)

0.25

7.25*10^-3

7.25*10^-50.2

0.244–0.253

D2

262–276

Peak(P-P)

0.255

8.65*10^-3

8.25*10^-50.14

0.246–0.265

D2

276–297

Peak(P-P)

0.251

8.25*10^-3

7.25*10^-50.880.240–0.258

D2

297–318

Peak(P-P)

0.25

7.25*10^-3

8.59*10^-50.14

0.242–0.264

D2

262–318

Peak(P-P)

0.251

9.25*10^-3

9.12*10^-50.87

0.244–0.262

D3

278

Absorbance (p-o)

0.254

8.32*10^-3

9.02*10^-50.74

0.246–0.265

D3

296

Absorbance (p-o)

0.2538.21*10^-3

8.25*10^-50.62

0.242–0.263

D3

311

Absorbance (p-o)

0.253

7.98*10^-3

8.75*10^-50.80.243–0.262

D3

267–287

Absorbance (p-o)

0.251

8.54*10^-3

8.85*10^-50.25

0.249–0.267

D3

287–306

Peak(P-P)

0.252

8.23*10^-3

7.65*10^-50.64

0.245–0.264

D3

306–317

Peak(P-P)

0.253

7.22*10^-3

8.15*10^-50.77

0.245–0.264

D3

267–317

Peak(P-P)

0.253

8.15*10^-3

9.14*10^-50.92

0.242–0.263

D4

270

Peak(P-P)

0.254

8.99*10^-3

9.45*10^-50.35

0.242–0.262

D4

287

Peak(P-P)

0.252

5.01*10^-3

5.45*10^-50.61

0.244–0.263

wavelength (287) nm were used to determine the effectof specific pharmaceutical additives on levofloxacin. The
estimation of levofloxacin at10 μg/mL does not exhibit
any additive effect, as indicated by the fourth derivativeof the peak absorbance measurement. The outcomes are
displayed in Table 6.
First derivative of peak absorbance measurement atwavelength (291) nm, second derivative of peak absorbance measurement at wavelength (291) nm, third derivative of peak absorbance measurement at wavelength(291) nm and fourth derivative of peak absorbance measurement at wavelength (291) nm were used to determine
the effect of specific pharmaceutical additives on norfloxacin. The estimation of Norfloxacin at 10 μg/mL does
not exhibit any additive effect, as indicated by the fourth
derivative of the peak absorbance measurement. The outcomes are displayed in Table 7.
First derivative of peak absorbance measurement atwavelength (294) nm, second derivative of peak absorbance measurement at wavelength (294) nm, third derivative of peak absorbance measurement at wavelength(294) nm and fourth derivative of peak absorbancemeasurement at wavelength (294) nm were used todetermine the effect of specific pharmaceutical additives
on moxifloxacin. Estimation of moxifloxacin at 10 μg/mL
does not exhibit any additive effect, as indicated by the
fourth derivative of the peak absorbance measurement.
The outcomes are displayed in Table 8.
Forced degradation study

The stress test findings for three antibiotics—levofloxacin, norfloxacin, and moxifloxacin—are displayed in thetable. In order to observe how drugs degrade, the test
subjects them to more severe conditions than they usually encounter. The most stable appears to be levofloxacin. In most cases, levofloxacin degrades more slowly
than norfloxacin. Similar to norfloxacin, moxifloxacin
breaks down a little bit more slowly (Table 9).
Figure 2 aims to illustrate the absorption characteristics of Levofloxacin, Norfloxacin, and Moxifloxacin. By
comparing these spectra, can potentially. Identify anddifferentiate between the three compounds based on
their unique peak patterns, gain insights into the structural similarities and differences between the molecules,

Elgendy et al. BMC Chemistry(2024) 18:115

Page 16 of 21

Table 12 Statistical evaluation of the elaborated method for norfloxacin (standard solution n = 10)
Derivative

λ (nm)

TechniqueD1

278

Absorbance (p-o)

y = 0.002x–0.0092

0.948

D1

300

Absorbance (p-o)

y = 0.0016x + 0.0103

0.997

D1

316–286

Peak(P-P)

y = 0.0359x–0.0199

0.941

D1

257–286

Peak(P-P)

y = 0.027x + 0.1501

0.990

D1

257–316

Peak(P-P)

y = 0.0134x–0.014

0.970

D2

310

Absorbance (p-o)

y = 0.0002x + 0.0006

0.979

D2

291

Absorbance (p-o)

y = 0.0004x–0.0014

0.974

D2

270

Absorbance (p-o)

y = 0.0002x + 0.0006

0.995

D2

254–279

Peak(P-P)

y = 0.0003x + 0.014

0.977

D2

279–298

Peak(P-P)

y = 0.0032x–0.015

0.985

D2

298–337

Peak(P-P)

y = 0.0042x + 0.0179

0.960

D2

254–337

Peak(P-P)

y = 0.0041x + 0.0163

0.962

D3

262

Absorbance (p-o)

y = 0.005x–0.002

0.994

D3

282

Absorbance (p-o)

y = 0.0003x + 0.0001

0.992

D3

311

Absorbance (p-o)

y = 0.0005x–0.0005

0.966

D3

297

Absorbance (p-o)

y = 0.0005x + 0.0002

0.985

D3

270–291

Peak(P-P)

y = − 0.0005x–0.0023

0.962

D3

253–270

Peak(P-P)

y = 0.0016x + 0.0003

0.984

D3

291–309

Peak(P-P)

y = 0.0008x + 0.0022

0.989

D3

309–337

Peak(P-P)

y = − 0.0001x–0.0005

0.997

D3

253–337

Peak(P-P)

y = 0.0006x + 0.0012

0.995

D4

267

Absorbance (p-o)

y = 0.027x + 0.1501

0.997

D4

258

Absorbance (p-o)

y = 0.0134x–0.014

0.951

D4

304

Absorbance (p-o)

y = 0.0002x + 0.0006

0.998

D4

321

Absorbance (p-o)

y = 0.0004x–0.0014

0.997

D4

291

Absorbance (p-o)

y = 0.0002x + 0.0006

0.985

D4

263–282

Peak(P-P)

y = 0.0003x + 0.014

0.998

D4

249–363

Peak(P-P)

y = 0.0032x-0.015

0.995

D4

297–310

Peak(P-P)

y = 0.0042x + 0.0179

0.998

D4

282–297

Peak(P-P)

y = 0.0041x + 0.0163

0.990

D4

310–319

Peak(P-P)

y = 0.005x-0.002

0.997

D4

249–319

Peak(P-P)

y = 0.0003x + 0.0001

0.989

provide a reference for further analysis using techniques
derivative spectroscopy. In the “peak-peak” technique,amplitude measurements (from maximum to minimum)
were conducted, while the baseline-to-peak techniqueinvolved measurements from the maximum to the zeroline or from the minimum to the zero line (Additional
file 1).
Figure 3 presents the first (1st), second (2nd), third(3rd), and fourth (4th) derivative spectra of levofloxacin. Each spectrum exhibits distinct features comparedto the original spectrum and other derivatives. The purpose of these derivative spectra is to enhance specificRegression equation

Correlation
coefficientcharacteristics and potentially improve the analysis of
levofloxacin.
Figure 4 presents the first (1st), second (2nd), third(3rd), and fourth (4th) derivative spectra of norfloxacin.
Each spectrum exhibits distinct features compared to theoriginal spectrum and other derivatives. The purpose of
these derivative spectra is to enhance specific characteristics and potentially improve the analysis of norfloxacin.
Figure 5 presents the first (1st), second (2nd), third(3rd), and fourth (4th) derivative spectra of moxifloxacin. Each spectrum exhibits distinct features comparedto the original spectrum and other derivatives. The purpose of these derivative spectra is to enhance specificElgendy et al. BMC Chemistry(2024) 18:115

Page 17 of 21

Table 13 Statistical analysis of the determination of norfloxacin in tablets “Epinor 400 mg” (n = 10)
Derivative

λ (nm)

TechniqueContent of
norfloxacin (g)Standard deviation

Variance

Correlation
coefficient

Confidence
interval
(95%)

D1

278

Absorbance (p-o)

0.402

9.67*10^-3

9.05*10^-50.81

0.343–0.464

D1

300

Absorbance (p-o)

0.405

6.20*10^-3

9.66*10^-50.41

0.395–0.421

D1

316–286

Peak(P-P)

0.401

8.25*10^-3

9.23*10^-50.65

0.380–0.435

D1

257–286

Peak(P-P)

0.401

7.62*10^-3

9.56*10^-50.58

0.365–0.475

D1

257–316

Peak(P-P)

0.403

8.52*10^-3

7.25*10^-50.2

0.412–4.265

D2

310

Absorbance (p-o)

0.402

9.24*10^-3

8.21*10^-50.26

0.389–4.005

D2

291

Absorbance (p-o)

0.402

9.87*10^-3

7.75*10^-50.52

0.392–0.421

D2

270

Absorbance (p-o)

0.407

8.26*10^-3

7.95*10^-50.8

0.392–0.410

D2

254–279

Peak(P-P)

0.403

9.65*10^-3

9.15*10^-50.75

0.385–0.402

D2

279–298

Peak(P-P)

0.404

9.66*10^-3

8.89*10^-50.2

0.396–0.419

D2

298–337

Peak(P-P)

0.402

7.25*10^-3

7.12*10^-50.690.400–0.415

D2

254–337

Peak(P-P)

0.403

8.32*10^-3

7.95*10^-5

0.7

0.394–0.411

D3

262

Absorbance (p-o)

0.404

9.21*10^-3

9.14*10^-50.31

0.410–0.425

D3

282

Absorbance (p-o)

0.402

8.22*10^-3

9.05*10^-50.8

0.408–0.417

D3

311

Absorbance (p-o)

0.406

7.45*10^-3

9.14*10^-50.95

0.384–0.414

D3

297

Absorbance (p-o)

0.404

9.12*10^-3

7.45*10^-50.62

0.397–0.408

D3

270–291

Peak(P-P)

0.403

8.98*10^-3

9.32*10^-50.31

0.380–0.405

D3

253–270

Peak(P-P)

0.404

8.14*10^-3

9.65*10^-50.95

0.393–0.415

D3

291–309

Peak(P-P)

0.405

8.25*10^-3

8.32*10^-50.45

0.382–0.407

D3

309–337

Peak(P-P)

0.402

9.25*10^-3

9.68*10^-50.9

0.382–0.400

D3

253–337

Peak(P-P)

0.403

9.25*10^-3

9.68*10^-50.2

0.373–0.404

D4

267

Absorbance (p-o)

0.404

5.87*10^-3

5.21*10^-50.65

0.384–0.421

D4

258

Absorbance (p-o)

0.404

5.24*10^-3

5.54*10^-50.65

0.392–0.424

D4

304

Absorbance (p-o)

0.403

4.02*10^-3

4.70*10^-50.6

0.374–0.416

D4

321

Absorbance (p-o)

0.404

3.25*10^-3

3.25*10^-50.620.437–0.264

D4

292

Absorbance (p-o)

0.403

5.97*10^-3

5.35*10^-50.52

0.403–0.410

D4

263–282

Peak(P-P)

0.405

3.20*10^-3

3.14*10^-50.65

0.402–0.422

D4

249–363

Peak(P-P)

0.403

3.63*10^-3

3.25*10^-50.61

0.403–0.464

characteristics and potentially improve the analysis ofmoxifloxacin Additional file 1.
Statistical analysis

The data were meticulously subjected to statistical analysis, expressing results in terms of standard deviation(%SD) and relative standard deviation (%RSD). The calculated %RSD values, consistently below 1%, underscore
the remarkably high precision achieved by these methods (refer to Tables 10, 12, 14). This statistical assessment further solidifies the reliability and reproducibilityof the developed methods. The content determinationof Levofloxacin, Norfloxacin, and Moxifloxacin in market products closely aligned with label claims, ranging
from 98.05% to 99.47%, indicating the reliability of the
proposed method. RSD values were within the range of
0.16% to 0.92% (refer to Tables 11, 13, 15).

Potency assay tests of levofloxacin were performed by
the proposed method. According to USP 29 [38], levofloxacin tablets must contain 95–105% of the labeledamount of drug. The brand products met the standard criteria with the new analytical method (Table 11).
For Levofloxacin in Levonex tablets, the "peak-zero"technique for the fourth derivative spectra at 287.0 nm
yielded the best results (SD—5.01*10^-3, RSD—0.61%).
Potency assay tests of norfloxacin were performed by
the proposed method. According to USP 29 [38], norfloxacin tablets must contain 95–105% of the labeled amountof drug. The brand products met the standard criteriawith the new analytical method (Table 13). For Norfloxacin in Epinor 400 mg tablets showed optimal results withthe "peak-zero" and "peak-peak" techniques for the fourth
derivative spectra at all examined wavelengths (RSD
about 0.65%).

Elgendy et al. BMC Chemistry(2024) 18:115

Page 18 of 21

Table 14 Statistical evaluation of the elaborated method for moxifloxacin (standard solution n = 10)
Derivative

λ (nm)

TechniqueD1

281

Absorbance (p-o)

y = 0.0013x + 0.0241

0.972

D1

304

Absorbance (p-o)

y = − 0.0013x–0.0244

0.972

D1

292–326

Peak(P-P)

y = − 0.0222x–0.4592

0.979

D1

262–292

Peak(P-P)

y = 0.0206x + 0.3726

0.960

D1

262–326

Peak(P-P)

y = − 0.0048x–0.0615

0.994

D2

311

Absorbance (p-o)

y = 0.0001x + 0.0024

0.964

D2

294

Absorbance (p-o)

D2

271

Absorbance (p-o)

y = 0.00009x + 0.00173

0.978

D2

262–278

Peak(P-P)

y = 0.0005x + 0.0091

0.948

D2

278–304

Peak(P-P)

y = − 0.0035x–0.0654

0.948

D2

304–331

Peak(P-P)

y = 0.0012x + 0.0261

0.942

D2

262–331

Peak(P-P)

y = − 0.0017x–0.0322

0.967

D3

287

Absorbance (p-o)

y = − 0.00002x–0.00041

0.942

D3

302

Absorbance (p-o)

y = 0.00004x + 0.00073

0.946

D3

315

Absorbance (p-o)

y = − 0.00001x–0.00026

0.963

D3

263–295

Peak(P-P)

y = − 0.0005x–0.0093

0.964

D3

295–309

Peak(P-P)

y = 0.0008x + 0.0022

0.948

D3

309–332

Peak(P-P)

y = − 0.0002x–0.0031

0.970

D3

263–332

Peak(P-P)

y = − 0.0003x–0.0065

0.953

D4

271

Absorbance (p-o)

y = − 0.000001x–0.000027

0.998

D4

277

Absorbance (p-o)

y = − 0.000002x–0.000050

0.998

D4

294

Absorbance (p-o)

y = 0.000008x + 0.000174

0.956

D4

307

Absorbance (p-o)

D4

320

Absorbance (p-o)

y = 0.000001x + 0.000028

0.956

D4

266–273

Peak(P-P)

y = 0.000001x + 0.000009

0.990

D4

273–282

Peak(P-P)

y = − 0.000009x–0.000111

0.989

D4

282–302

Peak(P-P)

y = 0.00008x + 0.00160

0.957

D4

302–313

Peak(P-P)

y = − 0.00005x–0.00097

0.956

D4

313–332

Peak(P-P)

y = 0.00004x + 0.00028

0.968

Potency assay tests of moxifloxacin were performed by
the proposed method. According to USP 29 [38], Moxifloxacin tablets must contain 95–105% of the labeledamount of drug. The brand products met the standard
criteria with the new analytical method (Table 15). For
Moxifloxacin in Delmoxa tablets demonstrated superior
outcomes with the "peak-zero" technique for the second
derivative spectrum at wavelength 294 nm (SD—5.3210^3, RSD—0.91%) and the third derivative spectrum atwavelength 302 nm (SD—5.3210^-3, RSD—0.45%).
The table presents the statistical evaluation of thedetermination of Moxifloxacin content in "Delmoxa 400"
tablets using various derivatives and techniques.

Regression equationy = − 0.0002x–0.004

y = − 0.00001x–0.00016

Correlation
coefficient

0.960

0.940

These results are provided to assess the accuracy andefficiency of the proposed method used for the analysis of
Moxifloxacin in “Delmoxa 400” tablets.

Discussion
In summary, the proposed analytical method proves tobe a highly effective tool for the routine quality control
analysis of Levofloxacin, Norfloxacin, and Moxifloxacin
in both pure and pharmaceutical samples employing.
First-, second-, third-, and fourth-order UV derivative
spectroscopy, offers a straightforward, quick, sensitive,and direct approach for determining the analyzed drugs.
The method offers accuracy, precision, selectivity, andElgendy et al. BMC Chemistry(2024) 18:115

Page 19 of 21

Table 15 Statistical evaluation of moxifloxacin determination in “Delmoxa 400” tablets (n = 10)
Derivative

λ (nm)

TechniqueContent ofmoxifloxacin (g)Standard deviation

Variance

Correlation
coefficient

Confidence
interval
(95%)

D1

281

Absorbance (p-o)

0.48.67*10^-3

7.45*10^-50.31

0.4025–0.426

D1

304

Absorbance (p-o)

0.4019.68*10^-3

9.91*10^-50.25

0.395–0.405

D1

292–326

Peak(P-P)

0.4

8.85*10^-3

8.65*10^-50.53

0.393–0.428

D1

262–292

Peak(P-P)

0.4

7.78*10^-3

9.36*10^-50.52

0.387–0.405

D1

262–326

Peak(P-P)

0.4

9.56*10^-3

9.52*10^-50.9

0.394–0.411

D2

311

Absorbance (p-o)

0.4017.65*10^-3

9.85*10^-50.85

0.405—0.419

D2

294

Absorbance (p-o)

0.45.32*10^-3

5.65*10^-50.91

0.401–0.429

D2

271

Absorbance (p-o)

0.49.65*10^-3

9.55*10^-50.66

0.389–0.415

D2

262–278

Peak(P-P)

0.4

7.65*10^-3

8.65*10^-50.80.390–0.425

D2

278–304

Peak(P-P)

0.401

9.89*10^-3

9.80*10^-50.52

0.407–0.418

D2

304–331

Peak(P-P)

0.401

7.95*10^-3

8.65*10^-50.60.401–0.426

D2

262–331

Peak(P-P)

0.4

9.45*10^-3

8.25*10^-50.41

0.403–0.424

D3

287

Absorbance (p-o)

0.4017.69*10^-3

9.65*10^-50.32

0.388—0.425

D3

302

Absorbance (p-o)

0.4015.32*10^-3

4.65*10^-50.45

0.381–0.410

D3

315

Absorbance (p-o)

0.49.55*10^-3

9.65*10^-50.71

0.394–0.405

D3

263–295

Peak(P-P)

0.401

7.98*10^-3

9.59*10^-50.46

0.406–0.420

D3

295–309

Peak(P-P)

0.401

8.80*10^-3

9.51*10^-50.25

0.405–0.420

D3

309–332

Peak(P-P)

0.401

9.78*10^-3

8.45*10^-50.170.400–0.415

D3

263–332

Peak(P-P)

0.4

8.65*10^-3

9.65*10^-50.81

0.408–0.415

D4

271

Absorbance (p-o)

0.4019.54*10^-3

7.98*10^-50.15

0.390–0.420

D4

277

Absorbance (p-o)

0.4018.90*10^-3

9.25*10^-50.64

0.395–0.427

D4

294

Absorbance (p-o)

0.4017.45*10^-3

7.77*10^-50.92

0.389–0.423

D4

307

Absorbance (p-o)

0.4018.65*10^-3

8.77*10^-50.25

0.397–0.402

D4

320

Absorbance (p-o)

0.4018.77*10^-3

7.69*10^-50.15

0.398–0.412

D4

266–273

Peak(P-P)

0.4

9.45*10^-3

9.45*10^-50.22

0.371–0.420

D4

273–282

Peak(P-P)

0.4

8.22*10^-3

9.56*10^-50.36

0.399–0.417

D4

282–302

Peak(P-P)

0.4

9.46*10^-3

9.33*10^-50.55

0.387–0.408

D4

302–313

Peak(P-P)

0.402

9.52*10^-3

9.41*10^-50.470.400–0.415

D4

313–332

Peak(P-P)

0.4

8.74*10^-3

8.65*10^-50.16

0.398–0.401

ease of use, making it a valuable addition to the analytical
toolbox.
Utilizing the Lambda 15 spectrophotometric, spectra
derivatives can be stored in the computer memory, allowing the retrieval of derivative values at any marked pointon the recorded spectrum. Two graphical techniques,
namely “peak-zero” and “peak-peak,” were employed for
determining spectra derivatives.
In the “peak-peak” technique, amplitude measurements(from maximum to minimum) were conducted, while thebaseline-to-peak technique involved measurements fromthe maximum to the zero line or from the minimum tothe zero line.
Linear equations derived from regression analysis for
Levofloxacin, Norfloxacin, and Moxifloxacin are presented in Tables 10, 12, and 14, respectively. Statistical evaluation of the results for the determination offluoroquinolones in pharmaceutical dosage tablets is
illustrated in Tables 11, 13, and 15.

Conclusions
The proposed approach demonstrates sensitivity, accuracy, simplicity, precision, speed, and economy. The
results showed an outstanding mean recovery (99.20–
100.08%) and a good correlation value (R2 = 0.999).
The recommended derivative spectrophotometry
method was successfully validated in accordance withICH Q2B criteria by using UV spectral data that was
produced from the analysis of chemically deteriorated
samples using the recommended methodology. In

Elgendy et al. BMC Chemistry(2024) 18:115

physical–chemical research, derivative spectrophotometry proves to be an extremely useful tool as it allows
for the non-invasive extraction of information from thefundamental spectrum. Recovery investigations support the great accuracy of these techniques even further. The method is found to be precise and accurate,with a linear response within the given range and lower
LOD and LOQ values where acceptable, making it suitable for routine quality control analysis for levofloxacin,
Norfloxacin, and Moxifloxacin in both pure form andpharmaceutical samples.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13065-​024-​01193-4.
Additional file 1. Examples Spectra Used In The Research.
Acknowledgements
The author presents great thanks to the Eipico Company for supporting thisstudy.
Author contributions
Kh. Elgendy: Methodology, Validation, Conceptualization, Project administration, Supervision, Writing—review & editing. M. Zaky: Datacuration, Supervision, Conceptualization, Investigation. T. Alaa Eldin: Data curation, Software,
Methodology. S. Fadel: Methodology, Soft-ware, Data curation, Formal analysis,
Validation, Writing—originaldraft.
Funding
Open access funding provided by The Science, Technology & Innovation
Funding Authority (STDF) in cooperation with The Egyptian Knowledge Bank
(EKB).
Data availability
The datasets during and/or analysed during the current study available fromthe corresponding author on reasonable request.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interest.
Received: 29 November 2023 Accepted: 17 April 2024

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