Alqalam Journal of Medical and Applied Sciences.

2025;8(2):924-928
https://doi.org/10.54361/ajmas.258257

Original article

Development and Validation of a Simple UV-Vis Spectrophotometric

Method for the Quantitative Determination of Amoxicillin in
Pharmaceutical Dosage Forms
Gazala Ben-Hander1* , Hawa Mousa2 , Adel Alajtal2
1Department of Chemistry, Faculty of Science, Sirte University, Sirte, Libya
2Department of Chemistry, Faculty of Science, Misurata University, Misurata, Libya
Corresponding email. [email protected]

Abstract
A simple, sensitive, and inexpensive UV/visible spectrophotometric approach was developed and
validated for the quantitative measurement of amoxicillin (AMX) in pharmaceutical dosage forms.
The approach demonstrated excellent linearity (r² = 0.9994) across the concentration range of 1.0-
15.0 µg mL⁻¹ by measuring absorbance at 228 nm. The determined limits of detection and
quantification were established at 0.56 and 1.72 µg mL-1 for AMX, respectively. Values of relative
standard deviation (RSD) below 2% indicated remarkable repeatability and consistency; intra-day
and inter-day studies confirmed accuracy. Recovery tests conducted at three concentration levels
yielded results within the acceptable range of 94.15% to 108%, demonstrating the accuracy of the
method. The validated method was successfully used to analyze various commercial amoxicillin
products in tablet and capsule forms because all the tested samples met the required quality
standards. These findings demonstrate that the proposed spectrophotometric method is consistent
and adequate for routine quality monitoring of amoxicillin in pharmaceutical formulations.
Keywords: Amoxicillin, UV/Visible Spectrophotometric, Method Validation, Capsules, Tablets.

Introduction
Amoxicillin is a common beta-lactam antibiotic commonly used to treat gram-negative bacterial infections
[1, 2]. Similar to other antibiotics of its class, this medicine suppresses cell wall synthesis, resulting in
osmotic lysis. Antibiotics are among the most used pharmaceuticals globally and are extensively utilized in
both human and veterinary medicine, not only for the treatment or prevention of infections in humans and
animals but also for boosting animal growth [3, 4]. AMX serves as a primary antibiotic for addressing minor
respiratory infections and various other prevalent infections. Chemically, It is a semi-synthetic antibiotic of
the penicillin class, characterized by attaching a thiazolidine ring to a 𝛽-lactam ring [5, 6]. The chemical
structure of amoxicillin (2S,5R,6R)-6-[[(2R)-2-amino-2-(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-
4-thia-1-azabicyclo[3.2.0]heptane-2-carboxylic acid (Fig. 1) [7]. It is an acid-stable and highly hygroscopic
white or slightly off-white powder [8], and is typically administered orally [9]. Due to its extensive bactericidal
efficacy and consequent prevalent application in pharmaceuticals, numerous formulations of this drug,
including capsules, tablets, oral suspension powder, injections, and combinations with other components
[10]. However, its use may also be associated with potential side effects include nausea, vomiting, rashes,
and antibiotic-associated colitis [11]. A minor change in the composition or purity of the active
pharmaceutical ingredient (API) can influence the therapeutic results and potentially lead to adverse effects
of medications. Consequently, it is essential to create enhanced analytical techniques for the pharmaceutical
evaluation of medications.
Several analytical methods have been developed to determine AMX, including thin-layer chromatography
(TLC) [1], high-performance thin-layer chromatography (HPTLC) [12, 13], high-performance liquid
chromatography (HPLC) [14-16], voltammetry[17, 18], electrochemical methods [19, 20], electrophoresis
[21], and spectrophotometric methods [22-25]. Among these, the spectrophotometric technique is the most
widely used method because of its simplicity and ease of use and because it does not require expensive
equipment. This study intends to develop a validated approach that is straightforward, efficient, and
economical for the quantitative assessment of amoxicillin in pharmaceutical tablet formulations via UV-Vis
spectrophotometric analysis.

Figure 1. The chemical structure of amoxicillin.

Copyright Author (s) 2025. Distributed under Creative Commons CC-BY 4.0
Received: 18-03-2025 - Accepted: 13-05-2024 - Published: 21-05-2025 924
 Alqalam Journal of Medical and Applied Sciences. 2025;8(2):924-928
https://doi.org/10.54361/ajmas.258257

Materials and methods

Equipment and materials
All spectral scans and measurements were done using a JENWAY 6305 spectrophotometer. We used
analytical reagent-grade chemicals throughout this study. Reference standard of pure AMX was purchased
from Sigma Aldrich. Two brands of AMX (Amoxicillin Tablets USP, Kwality, India and Ronakamoxi Capsules,
Fugen, India) were purchased from a local pharmacy store. Each of them contained 500 mg per the label
claim.

Standard preparation
Amoxicillin Solution Preparation
Standard amoxicillin stock solution (1000 μg mL⁻¹) was prepared by dissolving 100 mg in 100 mL (20:30)
(V/V MeOH:water) and was stored at 4ºC until the analysis.

Test preparation
Ten powdered capsules, each containing 500 mg of AMX, or ten tablets of amoxicillin, also 500 mg each,
from each product were weighed individually to determine the average weight of a tablet and subsequently
ground into a fine powder using a mortar. A specific amount of powder equivalent to 10 mg of amoxicillin
was accurately measured, transferred into a 100 mL volumetric flask, dissolved in a diluted solution, and
subjected to sonication for 10 minutes to improve solubility. The solution was subsequently diluted to a
final volume of 100 mL using the same diluted solution. The sample underwent filtration through Whatman
filter paper no. 41 to remove solid particulates, resulting in a stock solution with a concentration of 100 µg
mL-1. A solution of 10 μg mL⁻¹ was subsequently prepared and subjected to UV analysis. Each medicinal
substance was subjected to three replicates.

Results and Discussion

Validation of Analytical Method
Linearity, limit of detection (LOD), and limit of quantification (LOQ)
Constructing a calibration curve using a total of eight standard solutions allowed the assessment of the
linearity of the suggested method. The linearity of the calibration plots was evaluated using standard AMX
to cover the concentration range of 1.0-15.0 μg mL⁻¹. The calibration curve was established by plotting the
absorption (y) axis versus the AMX concentration (x) axis. The obtained calibration curve was linear over the
studied concentration range. The data analysis revealed a regression equation of y = 0.0274x - 0.0035 with
a correlation coefficient of r² = 0.9994, indicating excellent linearity due to its high r² value (Fig. 2). The limit
of detection (LOD) and limit of quantification (LOQ) were calculated using the following equations: "LOD =
3.3S/K" and "LOQ = 10S/K", where S is the standard deviation of the intercept and K is the slope of the
calibration curve. The LOD and LOQ were determined to be 0.56 μg mL⁻¹ and 1.72 μg mL⁻¹, respectively.
These results turned out to be an improvement over the previously reported limits of detection using various
methods like standard UV-Visible spectrophotometry [22]. HPLC coupled with UV detection [26], and even
the more advanced ratio-first derivative zero-crossing UV-Vis technique [27].

Table 1. Analytical parameters obtained from the calibration graph of amoxicillin determined by
the proposed spectrophotometric method.
Parameter Value
λmax 228
Linear range (µg mL )-1 1-15
LOD (µg mL-1) 0.56
LOQ (µg mL-1) 1.72
Slope 0.0274
Intercept 0.0035
Correlation coefficient (r2) 0.9994

Copyright Author (s) 2025. Distributed under Creative Commons CC-BY 4.0
Received: 18-03-2025 - Accepted: 13-05-2024 - Published: 21-05-2025 925
 Alqalam Journal of Medical and Applied Sciences. 2025;8(2):924-928
https://doi.org/10.54361/ajmas.258257

0.5
y = 0.0274x - 0.0035
0.4 R² = 0.9994

Absorption
0.3

0.2

0.1

0
0 2 4 6 8 10 12 14 16
Concentration μgmL-1

Figure 2. Calibration curve of amoxicillin.

Precision
The term "precision" denotes the extent to which measured values align with reference values. Precision is
generally contingent upon the analyte concentration; thus, it is essential to ascertain this concentration
while operating within the relevant range of interest. Repeatability (within-day precision) and intermediate
precision (between-day precision) were assessed over three consecutive days to evaluate overall precision
[28]. Three standard solution preparations were used to check the precision within a single day at three
different concentration levels (3, 7, and 9 μg mL⁻¹) with a total of nine measurements. Each concentration
was measured three times, and the inter-day precision was evaluated over three consecutive days (n = 27).
Table 2 shows that the calculated coefficient of variation for repeatability, intra-day, and inter-day results
stayed within the acceptable range, with RSD% not going over 2% [29]. Interestingly, the results were quite
similar to those obtained with previously reported methods, like flow injection paired with UV-Vis [30], or
standard UV-Vis spectroscopy [31]. The outcomes show that the procedure is very repeatable. A high degree
of precision was shown by sufficiently low coefficients of variation. Therefore, the established approach may
be used reliably for determining AMX in pharmaceutical formulations.

Table 2. Amoxicillin standard solution's intra-day and inter-day precision (%RSD).

AMX concentration (μg mL-1) RSD (%)
Intra-day precision (n=9) 0hr 1hr 2hr
5 0.71 1.38 1.12
7 0.88 0.93 1.02
9 0.95 1.14 1.29
Inter-day precision (n=27) 1st day 2nd day 3rd day
5 1.94 1.87 1.40
7 1.98 1.22 0.88
9 1.34 1.04 1.57

Recovery
An analytical method's accuracy can be described as the degree to which the result of the value found is
close to the reference value [28]. This is a relatively close relationship between the two values. To ascertain
the precision of the proposed method, recovery experiments were carried out at three distinct
concentrations, namely 3, 7, and 9 μg mL⁻¹. The recoveries were carried out by including determined
quantities of AMX into the formulations that had been pretreated. A triplicate of each concentration level
was generated, and it was then subjected to UV/Visible three times. Table 3 summarizes the collected
results. This indicates that this method has the potential to determine the AMX in tablet dosage
formulations, as the outcome demonstrated that the approach was accurate. At each additional
concentration, the spiked drug yielded the best recoveries, ranging from 94.15 to 108 percent. This falls
within the recommended recovery percentage range of 80 to 110% [32].

Table 3. Recovery of the proposed method.

Sample (μg mL-1) Added (μg mL-1) Found (μg mL-1) Recovery ± RSD(%)*
7 3 10.26 108±0.44
7 7 13.59 94.15±0.36
7 9 16.9 97.11±0.044

Analysis of pharmaceutical formulation

The validated method was successfully employed for the testing of two commercial items of different types
(tablet and capsule) containing 500 mg of AMX. The findings are derived from the average of three specified

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Received: 18-03-2025 - Accepted: 13-05-2024 - Published: 21-05-2025 926
 Alqalam Journal of Medical and Applied Sciences. 2025;8(2):924-928
https://doi.org/10.54361/ajmas.258257

values presented in Table 4. The result indicates that there were no major differences between the amoxicillin
amounts listed by the manufacturer and those measured by the new method, which needed to be between
90% and 110% of the labeled amount [33]. Consequently, all brands examined adhered to USP
pharmacopeial standards.

Table 4. Assay results of amoxicillin in different pharmaceutical formulation.

Amount of drug Amount of drug % Labelled
Brand
labelled (mg) estimated (mg) claim±RSD
Amoxicillin Tablets USP (tablet) 500 513.0 102.6±0.442
Ronakamoxi (capsules) 500 553.5 110.7±0.598

Conclusion
The validation of an analytical method based on UV/visible spectroscopy was applied successfully for the
determination of amoxicillin in two pharmaceutical dosage forms, including tablets and capsules. The
proposed method was found to be linear within the concentration range of 1.0–15.0 μg mL⁻¹ with a
correlation coefficient (r² = 0.9994). The method demonstrated good sensitivity, evidenced by the low LOD
and LOQ values. Precision studies have validated that the method demonstrates exceptional repeatability
and reproducibility, with %RSD values consistently remaining under 2%. Accuracy was supported by
recovery rates ranging from 94.15% to 108%, which fall within acceptable limits. Furthermore, analysis of
commercial amoxicillin products indicated that all tested brands met pharmacopeial standards. The good
recoveries and low coefficients of variation that are obtained make the suggested method suitable for the
routine quality control analysis of amoxicillin in pharmaceutical dosage formulations.

Acknowledgments
The authors would like to thank Misurata University for its support.

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Copyright Author (s) 2025. Distributed under Creative Commons CC-BY 4.0
Received: 18-03-2025 - Accepted: 13-05-2024 - Published: 21-05-2025 928