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Validation of Dissolution Method of Levofloxacin 750 mg Tablet by High
Performance Liquid Chromatography
Mithun Chandra Banik*
1
, Professor Dr. Bishyajit Kumar Biswas
2
, Bhorhan Uddin Sawan
3
, Sanjit
Biswas
4
, Dr. Md Abu Taib Al Refat
5
1 2
Department of Pharmacy, Jagannath University, Dhaka, Bangladesh,
3
Department of Pharmacy, Noakhali Science and Technology University
4
Department of Pharmacy, Khulna University
5
MBBS (Dhaka National Medical College, Dhaka, Bangladesh), Mph (Lincoln University College,
Malaysia)
*Corresponding author
DOI: https://dx.doi.org/10.51244/IJRSI.2025.1210000155
Received: 06 October 2025; Accepted: 14 October 2025; Published: 12 November 2025
ABSTRACT
Dissolution test is needed to investigate the drug release of dosage form and its performance in vivo. The lot-
to-lot quality of drug product is estimated by dissolution test. Research and validation of procedures of
dissolution is of utmost significance during formation of new formulation and quality control. The process of
dissolution should be adequately formulated and justified. This paper aims to summarize the creation and
validation of dissolution procedure/s as well as to propose a feasible method of specificity, linearity, range,
accuracy, and precision of the methods and limit of detection and limit of quantitation. Validation and
development of dissolution test procedures may be an unwieldy process, in several dimensions. Techniques
need to be generated and proven not only of the dissolution test technique per se, but also of some assay to
determine the test outcomes [1]. This research aims at establishing, testing, and testing a strong dissolution
process of Levofloxacin 750 mg tablets, using RP HPLC. The dissolution process will provide a reflection of
what happens in vivo when administering the drug and assist in evaluating quality on a lot-to-lot basis and in
the comparability of generic and brand-name products [2].
Key Words: Levofloxacin, HPLC, Assay, Method Validation, Dissolution, Chromatography
Levofloxacin:
Levofloxacin is an antimicrobial agent. It is administered in the treatment of several bacterial infections. It may
be used as well to treat tuberculosis, meningitis, pelvic inflammatory diseases among some of its other uses
Fig 1: Structure of Levofloxacin
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Its use is not suggested as a rule of thumb under the circumstances when alternative services are available. It is
administered through the mouth, intravenously and as eye drops. The side effects are very common, they are
nausea, diarrhea, disturbing sleep. Tendon rupture, tendon inflammation, seizure, psychosis and possibly
irreversible damage to peripheral nerves are potentially severe side effects. The damage on the tendons can be
seen months after the treatment. Individuals can also get sun burned much easier. Myasthenia gravis may
further deteriorate in terms of muscle weakness in affected people and difficulty with breathing. Although it
was not suggested to be used during pregnancy, there seems to be low risk. Use of other drugs under this group
seems to be safe during breast feeding, but the safety of the Levofloxacin is still uncertain. Levofloxacin is a
general antibiotic belonging to the group of drugs obtained using the fluroquinolone. It normally leads to death
of the bacteria. It is an isomer of the medicine ofloxacin which is left sided.
Levofloxacin was invented in 1985 and licensed to treatment in the US in 1996. It is available as generic drug.
In the third world countries, the wholesale treatment costs approximately 0.44- 0.95 a week [3].
Approximately, a week of treatment in the US costs between 50-100. As of 2016 it ranked as the 161 to be
prescribed most in the United States with over 3 million prescription numbers.
Aim:
Validation of dissolution procedure aims at proving that it is appropriate to the purpose of its implementation.
Dissolution validation is relevantly significant in the role of developing pharmaceutical [4].
The aim and goal included to come up with a dissolution method to be used in the routine analysis of
Levofloxacin 750mg tablet with RP-HPLC to analyze it. It is conducted to derive data that have potential in
high quality, peak efficacy and safety of drug therapy in addition to peak economy in drug production [5]..
Objectives:
To know the validation parameters of Levofloxacin that includes System Precision, Method Precision,
Linearity [6].
To know about the drug Levofloxacin in shortly.
To minimize validation method and instrumental error.
Assure consistent production performance.
To give reliable and reproducible results in accordance with the given specification of the test method
[7].
To ensure the quality of test results.
To assured of the correctness of results.
To minimize rejection loss.
Help timely corrective action.
Reduce extensive and product testing.
The scope is the application of the validated methods for routine analysis which may be included in the
pharmaceutical monograph in future.
LITERATURE REVIEW
Journal Name
International Journal of Advances in Pharmacy, Biology and Chemistry
Author Name
K. Srinivas
Method Name
RP-HPLC Method
Column Name
Phenomenex column 250 x 4.6 mm, 5μm
Mobile Phase
Orthophosphoric acid: Methanol (70:30)
Wave Length
230 nm
Run Time
30 min
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Flow Rate
1 ml /min
Journal Name
International Journal of Pharmaceutical and Clinical Research
Author Name
Shafrose Syed, Haritha Pavani
Method Name
Validated Simultaneous Estimation and Development of Levofloxacin.
Column Name
Hypersil BDS C18 Column
Mobile Phase
Acetonitrile (75:25)
Wave Length
315 nm
Run Time
8 min
Flow Rate
1 ml /min
Journal Name
International Journal of Pharmacy and Pharmaceutical analysis
Author Name
Vaddeswarapu Madhavi and Mandalapu Neehanika
Method Name
RP-HPLC Method
Column Name
Intersil ODS C18 column
Mobile Phase
Methanol
Wave Length
223 nm
Run Time
6 min
Flow Rate
1 ml /min
Journal Name
International Journal of Chemical Science
Author Name
CH. Narasimha Raju Bh,
Method Name
RP-HPLC Method
Column Name
Phenomenex column 250 x 4.6 mm, 5μm
Mobile Phase
Methanol
Wave Length
295 nm
Run Time
110 min
Flow Rate
1 ml /min
Journal Name
Research Gate Journal
Author Name
E Reddy, Raghuram Reddy
Method Name
Development and Validation of Levofloxacin
Column Name
Hypersil BDS C18 Column 120 A (250 x 4.6mm), 5μm
Mobile Phase
Acetonitrile and Methanol (60:40)
Wave Length
285 nm
Run Time
5 min and 3 min
Flow Rate
1 ml /min
Materials and Reagents:
The reference standard of Levofloxacin Hemihydrate (Potency 98.0%) was obtained from Eskayef
Pharmaceutical Limited, Tongi, Gazipur 1711, Bangladesh and Levofloxacin (Trevox 750 mg and Levox 750
mg) tablets manufactured by Square Pharmaceuticals Limited and Opsonin Pharmaceutical Limited
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respectably were purchased from local pharmaceutical market (Table 1). HPLC grade acetonitrile from Korea,
HPLC grade water from Thiland and Trifluoroacetic acid was obtained from Korea.
Table1. Lists of Materials
Material
Manufacturer
Levofloxacin Hemihydrate (Standard)
Eskayef Pharmaceutical Limited
Levofloxacin (Trevox)
Square Pharmaceutical Limited
Levofloxacin (Levox)
Opsonin Pharmaceutical Limited
Table 2. Lists of Reagents
Reagents
Manufacturer
Acetonitrile
Daejung Chemical Co., Korea
Water
RCI Labscan Ltd., Thailand
Trifluoroacetic acid
Samchun Pure Chemical Co., Korea
Instrumentation:
Chromatographic analysis was performed on Adept series CECIL [8] (Cecil Instruments Ltd., Cambridge, UK)
with power Stream Software in RP-HPLC (Fig-2). Adept CECIL system CE 4900 was equipped with 4102
gradient pump, CE 4300 UV detector and CE 4020 degasser. AGE injector from Australia was used for
manual injection (Fig-3), Dissolution Tester RC-6 (Fig-4) from Korea was used.
Table 3. Lists of Instruments
Instruments
Source
HPLC
Cecil Instruments Ltd., Cambridge, UK
Dissolution Tester
Humanlab Instrument Co. Korea
Ultrasonic Cleaner
Spectrolab (England)
Electronic Balance
Thomas Scientific
Fig. 2: HPLC
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Fig.3 - Microinjection
Fig. 4 - Dissolution Tester
Fig.5 - Ultrasonic
Fig.6 - Electronic Balance
Chromatographic Condition:
Column: An analytical reversed phase column C8 was used for analysis. Chromatographic was performed in
ambient temperature.
Flow Rate: 0.3 ml/min
Detection: 295 nm
Runtime: 25 min
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Mobile Phase: Acetonitrile and Water with 0.15% Trifluoroacetic Acid (buffer). Mobile phase used in the
following ratio (Table 4)
Table 4: Gradient Condition in Mobile Phase
Time (min)
Mobile Phase A (%)
Mobile Phase B (%)
0
10
90
5
20
80
8
30
70
10
40
60
12
50
50
15
40
60
20
30
70
22
20
80
25
10
90
Preparation of mobile phase:
Mobile Phase- A:
Mobile phase A was prepared by adding 0.15% of Trifluoroacetic acid in 1L Acetonitrile.
Mobile Phase- B:
Mobile phase B was prepared by adding 0.15% of Trifluoroacetic acid in 1L Water.
Preparation of Blank:
86.9 ml conc. HCl is added in 10L water and 0.1N HCl was prepared.
Preparation of Standard:
The reference standard of Levofloxacin Hemihydrate 100 mg was weighted and taken in 100ml flask. After
10ml take from solution into another volumetric flask and make it 100ml. Therefore, per ml solution contains
100μg/ml [9].
Preparation of Sample:
Take 900 ml 0.1N HCl in dissolution tester 6 vessels [10]. Levofloxacin 750 mg 6 tablets (Trevox) and
Levofloxacin 750 mg 6 tablets (Levox) were given to 6 vessels. So, conc. will be 1.16 mg/ml. After 30 min
and 45 min 10 ml sample is collected from each vessel. After filtration 10 ml is taken and volume up to 50 ml
with 0.1N HCl. So, conc. will be now 116 μg/ml [10].
Method Validation:
Method validation is the “process of establishing documented evidence” which provides a high degree of
assurance [11]. The method was validated as per ICS guidelines. The method was validated by performing:
Parameters
Dissolution
System Precision
+
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Method Precision
+
Linearity
+
System Precision:
The system precision is checked by using standard chemical substance to ensure that the analytical system is
working properly. [12].
Method Precision:
In method Precision, a homogenous sample of single batch should be analyzed 6 times. [13].
Linearity:
Linearity is the property of a mathematical relationship or function which means that it can be graphically
represented as a straight line [14]. In analytically it directly proportional to the concentration of the analyte and
sample. [15]
From stock solution 1, pipette out 6ml, 8ml, 10ml, 12ml, 14ml into 100 ml volumetric flask to get 60% 80%
100% 120% 140% of standard solution [16].
System Precision:
System Precision was determined by repeatability of the Levofloxacin hemihydrate standard solution [17]. The
system precision of the method was evaluated by carrying out six determinations [18]. The areas of all
injections were taken from Power Stream (Table-5); mean, standard deviation and % relative standard
deviation was calculated. The calculation formula:
Standard deviation
% RSD = --------------------------------------------- x 100
Mean
Table 5: System Precision Data
Parameters
Data
Number of determinations(n)
6
Individual results (areas)
9373.3, 9355.2, 9386.7, 9402.3, 9397.2, 9483.5
Mean (area)
9399.7
Standard deviation (SD)
47.55
%RSD
0.51
Acceptance Criteria
Co-efficient of variation NMT 2.0%
*NMT = Not More Than
Result and Evaluation:
The system was precise, as the co-efficient of variation was less than 2.0%.
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Method Precision:
Dissolution sample Preparation:
10 ml Dissolution media containing drug was withdrawn from dissolution vessel after 30 minutes [19]. Then
from this 7 ml was diluted to 50 ml. Then it was filtered and injected.
Table 6: Method Precision Data
Parameters
Data
Number of determinations(n)
6
Individual dissolution results (%)
92.18, 93.40, 96.03, 93.92, 91.60, 92.69
Mean
93.30
Standard deviation (SD)
1.57
Relative Standard Deviation (%RSD)
1.68
Acceptance Criteria
Co-efficient of variation NMT 2.0%
Result and Evaluation:
The method was precise, as the co-efficient of variation was less than 2.0%.
Figure 7: Blank chromatogram of Levofloxacin Dissolution
Naturally, the Figure 7 shows the blank chromatogram of the Levofloxacin dissolution by the HPLC, there is
only one major peak, with large absorbance at the 4-minute mark and then with stable position at the end of 6
minutes without any notable peaks. This shows little interference or impurities in the blank sample.
Figure 8: Standard chromatogram of Levofloxacin Dissolution
The standard chromatogram of the dissolution of Levofloxacin presented in Figure 8 indicates that there was a
prominent peak at about 6.3 minutes whose absorbance was high to a level of about 450 mAU. This significant
peak indicates Levofloxacin and the height should be constant and clear after this peak which proves the
successful separation and purity of the standard solution.
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Linearity:
The linearity of an analytical procedure is its ability (within a given range) to obtain test results which are
directly proportional to the concentration of analyte in the sample.
Linearity with Standard Solution:
The linearity of peak area (y)/ concentration (x) of a dilution series was determined for standard solution in the
range 60% - 140% (e.g. 60,80,100,120,140) % of the nominal concentration.
Table 7: Linearity Data
Concentration (µg/ml)
Area (mAs)
60
5553.8
80
7514.4
100
9373.3
120
11347.6
140
13310.7
Figure 9: Levofloxacin Dissolution Linearity curve (60% -140%)
In Figure 9 it can be observed the Levofloxacin Dissolution Linearity at the concentration level of 60 percent
to 140 percent. In the graph the regression equation was y = 94.387x and the correlation coefficient (R2) =
0.9999 showing that there was high linearity between concentration (ug/ml) and peak area (mAs). The findings
affirm that the procedure is accurate and gives reliable consistent quantification over the concentration range
that was tested.
Acceptance Criteria:
Correlation Coefficient, r2 is ≥ 0.999
Result and Evaluation:
The Correlation Coefficient, r2 was found 0.9993. This method is sufficient linear as the Correlation
Coefficient, r2 is ≥ 0.999.
Range:
To determine the range of the method, 60% solution for 6 replicas and 140% solution for 6 replicas were
injected and %RSD was calculated.
5553.8
7514.4
9373.3
11347.6
13310.7
y = 94.387x
R² = 0.9993
0
2000
4000
6000
8000
10000
12000
14000
0 50 100 150
Area (mAs)
Conc (µg/ ml)
Linearity
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Table 8: Data of Range
Ranges
Areas (mAs)
Average
Standard Deviation
(SD)
% RSD
Remarks
60%
5553.8, 5592.3, 5409.5,
5529.7, 5486.0, 5543.4
5519.12
63.83
1.16
Complies
140%
13310.7, 13555.2, 13375.4,
13597.3, 13611.4, 13768.7
13536.45
167.64
1.24
Complies
Acceptance Criteria:
Method is suitable if co-efficient of variation is less than 2.0% for each level.
Result and Evaluation:
The method is suitable as %RSD is within limits.
Figure 10: Chromatogram of Levofloxacin at Range 60%
The chromatogram shown in Figure 10 is that of Levofloxacin at 60% concentration. The retention time of
6.23 is a sharp, symmetrical peak which shows good separation and detection. It has a peak area of 5553.8
mAs and a height of 287.1 mA which indicates the correct quantification of the analysis at this concentration
level.
Figure 11: Chromatogram of Levofloxacin at Range 140%
It is yet easy to understand through the presentation of Figure 11 that the chromatogram of the Levofloxacin at
concentration of 140 percent. At 6.32 minutes, there is a sharp and well-defined peak which has an excellent
resolution. its highest area is 13310.7 mAs and a height is 601.1 mA meaning omnipotent detector reaction and
excellent linearity at this degree of concentration.
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Percentage of Dissolution of Levofloxacin (Trevox) 750 mg Tablet After 30 minutes:
Standard Solution
Sample Solution
After 30 minutes
Replica
Area of Std (mAs)
Replica
Area (mAs)
Average
Area (mAs)
% of Dissolution
1
9373.3
Tab1
1
9967.0
10050.85
92.18
2
9355.2
2
10134.7
3
9386.7
Tab2
1
10234.2
10184.45
93.40
4
9402.3
2
10134.7
5
9397.2
1
10452.3
10470.45
96.03
6
9483.5
2
10488.6
Average
9399.7
1
10192.8
10240.75
93.92
2
10288.7
1
9953.2
9987.75
91.60
2
10022.3
1
10024.4
10106.35
92.69
2
10188.3
Average
93.30
Acceptance Criteria:
After 30 minutes dissolution should be ≥70%
Result and evaluation:
From the above table all tablets (6 tablets) comply the acceptance criteria.
Percentage of Dissolution of Levofloxacin (Trevox) 750 mg Tablet After 45 minutes:
Standard Solution
Sample Solution
After 45 minutes
Replica
Area of Std (mAs)
Replica
Area (mAs)
Average Area (mAs)
% of Dissolution
1
9373.3
Tab1
1
10220.9
10328.2
94.72
2
9355.2
2
10435.4
3
9386.7
Tab2
1
11044.3
11000.3
100.89
4
9402.3
2
10956.3
5
9397.2
Tab3
1
10753.7
10594.25
97.16
6
9483.5
2
10434.8
Average
9399.7
Tab4
1
10356.8
10472.65
96.05
2
10588.5
Tab5
1
10972.5
10917.4
100.13
2
10862.3
Tab6
1
11054.1
11064.75
101.48
2
11075.4
Average
98.40
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Acceptance Criteria:
After 45 minutes dissolution should be ≥75%
Result and evaluation:
From the above table all tablets (6 tablets) comply the acceptance criteria.
Figure 12: Standard chromatogram of Levofloxacin
The normal chromatogram of levofloxacin can be seen in figure 12 and indicates that there was sharp and
prominent peak at 6.29 minutes of retention time. High purity and correct resolution are also denoted by the
peak. Proper detection and quantification is attested by the area under the curve of 9373.3 mAs and a height of
440.8 mA.
Figure 13: Dissolution Chromatogram of Levofloxacin (Trevox) 750 mg Tablet after 30 minutes
Understandably, in the Figure 13, the dissolution chromatogram of the Levofloxacin (Trevox) 750 mg tablet
30 minutes later with a clear peak at a retention strength of 6.25 minutes. The sharp peak that has an area of
9967.0 mAs and the height of 515.0 mA allows attaining efficient drug release and a good chromatographic
resolution.
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Figure 14: Dissolution Chromatogram of Levofloxacin (Trevox) 750 mg Tablet after 45 minutes
Figure 14 depicts the dissolution chromatogram of Levofloxacin (Trevox) 750 mg tablet 45 minutes later and a
sharp and well-defined peak is observed in 6.33 minutes. An area of 10220.9 mAs and a height of 485.2 mA
appear in the chromatogram and it is a marker of successful dissolution and that the drug is released in a stable
way.
Percentage of Dissolution of Levofloxacin (Levox) 750 mg Tablet After 30 minutes:
Standard Solution
Sample Solution
After 30 minutes
Replica
Area of Std (mAs)
Replica
Area (mAs)
Average Area (mAs)
% of Dissolution
1
9373.3
Tab1
1
10291.2
10211.15
93.65
2
9355.2
2
10131.1
3
9386.7
Tab2
1
9978.5
9917.4
90.95
4
9402.3
2
9856.3
5
9397.2
Tab3
1
10176.5
10215.9
93.69
6
9483.5
2
10255.3
Average
9399.7
Tab4
1
9839.4
9858.45
90.41
2
9877.5
Tab5
1
10038.4
10071.25
92.37
2
10104.1
Tab6
1
9956.3
9971.9
91.45
2
9987.5
Average
92.09
Acceptance Criteria:
After 30 minutes dissolution should be ≥70%
Result and evaluation:
From the above table all tablets (6 tablets) comply the acceptance criteria.
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Percentage of Dissolution of Levofloxacin (Levox) 750 mg Tablet After 45 minutes:
Standard Solution
Sample
Solution
After 45
minutes
Replica
Area of Std
(mAs)
Replic
a
Area (mAs)
Average Area
(mAs)
% of Dissolution
1
9373.3
Tab1
1
11198.9
11122.1
102.00
2
9355.2
2
11045.2
3
9386.7
Tab2
1
10737.3
10720.1
98.32
4
9402.3
2
10702.9
5
9397.2
Tab3
1
10978.2
10926.85
100.21
6
9483.5
2
10875.5
Averag
e
9399.7
Tab4
1
10522.7
10531.05
96.58
2
10539.4
Tab5
1
10645.3
10577.35
97.01
2
10509.4
Tab6
1
10976.2
10922.35
100.17
2
10868.5
Average
99.05
Acceptance Criteria:
After 45 minutes dissolution should be ≥75%
Result and evaluation:
From the above table all tablets (6 tablets) comply the acceptance criteria.
Figure 15: Standard chromatogram of Levofloxacin
The Figured 15 indicate the standard chromatogram of Levofloxacin showing a sharp and high peak with a
retention of 6.29 minutes. The maximum value shows the presence, and purity of Levofloxacin, with an area of
9373.3 mAU*s and a height of 449.8 mAU, which proves correct separation of chromatography.
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Figure 16: Dissolution Chromatogram of Levofloxacin (Levox) 750 mg Tablet after 30 minutes
It can be well understood based on the figure 16 that the dissolution chromatogram of the Levofloxacin
(Levox) 750 mg tablet after 30 minutes which shows a sharp peak with a retention time of 6.38 minutes. The
chromatogram shows efficient drug release with the area of 10291.2 mAU+s and with the height of 445.1
mAU, which points to the coherent dissolution and performance of assays.
Figure 17: Dissolution Chromatogram of Levofloxacin (Levox) 750 mg Tablet after 45 minutes
The figure 17 is the dissolution chromatogram of Levofloxacin (Levox) 750 mg tablet at the end of 45 minutes
and it shows a sharp peak at 6.41 minutes. This chromatogram shows drug release was efficient with an area of
11198.9 mAU+s, height of 455.7 mAU, and indicates that the drug dissented well, and the assay was reliable.
CONCLUSION
The dissolution method for Levofloxacin 750 mg tablets, developed and validated in this study, demonstrated
rapid, sensitive, precise, and accurate performance [20]. These met the predefined acceptance criteria with low
variability (RSD well below 2%), and linearity showed excellent correlation (r^2 ≥ 0.999) over the tested range
[21]. The method produced consistent dissolution results at 30 and 45 minutes, with all tablets meeting the
specified dissolution criteria (≥70% at 30 minutes and ≥75% at 45 minutes). The range and robustness
assessments indicated suitability for routine quality control and comparability across brands. While stability
testing could not be completed due to time constraints, the current data support the method’s reliability for
routine analyses and potential inclusion in pharmaceutical monographs after additional stability evaluation.
Overall, the validated RP-HPLC dissolution procedure provides a practical, transferable approach for quality
assurance of Levofloxacin 750 mg tablets in Bangladesh and similar settings.
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INTERNATIONAL JOURNAL OF RESEARCH AND SCIENTIFIC INNOVATION (IJRSI)
ISSN No. 2321-2705 | DOI: 10.51244/IJRSI |Volume XII Issue X October 2025
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