INTRODUCTION:

Levofloxacin is a third-generation fluoroquinolone antibiotic commonly prescribed for the treatment of infections such as pneumonia, bronchitis, urinary tract and skin infections. It is the active L-isomer of ofloxacin and exhibits improved antibacterial activity along with favorable pharmacokinetic properties. Due to increasing bacterial resistance and variability in drug response, precise quantification of levofloxacine in pharmaceutical products and biological samples has become essential¹

Drug Profile:

IUPAC Name: (−) -(S)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methylpiperazin-1-yl)-7-oxo-7H-pyrido[1,2,3-de] [1,4] benzoxazine-6-carboxylic acid

Molecular Formula: C₁₈H₂₀FN₃O₄

Molecular Weight: 361.37g/mol

Figure 1: Structure of Levofloxacin⁴

Pharmacodynamics of Levofloxacin:

Levofloxacin exhibits concentration-dependent bactericidal activity. The effectiveness of the drug is primarily associated with pharmacodynamic parameters such as the ratio of peak plasma concentration (Cmax) to minimum inhibitory concentration (MIC) and the area under the curve (AUC/MIC). It is active against a broad range of Gram-positive and Gram-negative organisms.²

Pharmacokinetics of Levofloxacin:

After oral administration, levofloxacin is rapidly absorbed with nearly complete bioavailability. Peak plasma levels are typically achieved within 1-2 hours. The drug is widely distributed throughout body tissues and is mainly excreted unchanged via the kidneys.³

Mechanism of Action of Levofloxacin:

Levofloxacin exerts its antibacterial effect by inhibiting bacterial enzymes DNA gyrase (topoisomerase II) and topoisomerase IV. These enzymes are essential for DNA replication, transcription, and repair processes. Their inhibition disrupts DNA function, ultimately leading to bacterial cell death.

Figure 2: Mechanism of action of Levofloxacin

Analytical techniques in pharmaceutical analysis for determination of levofloxacin:

Various analytical approaches have been developed for the estimation of levofloxacin in bulk drugs, dosage forms, and biological samples.

UV–Visible Spectrophotometry:

Levofloxacin shows maximum absorbance in the range of 287-295nm. UV spectrophotometric techniques are widely used due to their simplicity, cost-effectiveness, and suitability for routine analysis. Methods such as derivative and simultaneous estimation have also been reported. 5

High-Performance Liquid Chromatography (HPLC):

HPLC is one of the most reliable and commonly used techniques for the quantification of levofloxacin. Reverse-phase HPLC methods using C18 columns and UV detection are frequently employed. These methods provide high accuracy, precision, and reproducibility, making them suitable for pharmaceutical and biological analysis. ⁶

High-Performance Thin Layer Chromatography (HPTLC):

HPTLC is a rapid and efficient technique that allows simultaneous analysis of multiple samples. It is commonly used for routine quality control due to its simplicity and relatively low cost. ⁷

LC–MS/MS (Mass Spectrometry):LC–MS/MS is a highly sensitive and selective technique for the analysis of levofloxacin in plasma and other biological matrices, whereby it has found application in pharmacokinetic and bioequivalence studies. Electrospray ionization has been particularly used for this purpose. ⁸

Fourier Transform Infrared Spectroscopy (FTIR):

FTIR spectroscopy was employed for identification and compatibility studies of drug–excipient interactions for the studied drug, levofloxacin.⁹

Nuclear Magnetic Resonance (NMR):

For the structural characterization and purity determination of drug substances, NMR spectroscopy has been applied to provide detailed molecular information on levofloxacin. However, NMR spectroscopy is less frequently employed for the quantification of this compound.¹⁰

Summary of Analytical Methods:

Table 1: Analytical methods development and validation for Levofloxacin single dosage forms by RP-HPLC

Sr.No.	Drug	Method	Description	References
1	Levofloxacin	Development and validation of RP-HPLC method for determination of Levofloxacin in tablets	RP-HPLC; Wavelength: 294nm; Column: C18 (250 × 4.6mm, 5µm); Mobile phase: Acetonitrile: Phosphate buffer (60:40 v/v); Flow rate: 1.0mL/min; Retention time: ~3.5min; Linearity: 2–20µg/mL; Correlation coefficient: 0.999	11
2	Levofloxacin	Stability indicating RP-HPLC method	RP-HPLC; Wavelength: 287nm; Column: C18; Mobile phase: Methanol: Water (70:30 v/v); Flow rate: 1.0mL/min; Linearity: 5–50µg/mL; Degradation studies performed	12
3	Levofloxacin	Bioanalytical RP-HPLC method in plasma	RP-HPLC; Wavelength: 295nm; Column: C18; Mobile phase: Acetonitrile: Buffer (65:35 v/v); Flow rate: 0.8mL/min; Linearity: 0.1–10µg/mL; High sensitivity	13

Table 2: Analytical methods development and validation for Levofloxacin by UV–Visible Spectrophotometry

Sr. No	Drug	Method	Description	References
1	Levofloxacin	UV spectrophotometric method	Wavelength: 287nm; Solvent: Distilled water; Linearity: 2–12µg/mL; Simple and rapid method	14
2	Levofloxacin	Derivative spectrophotometric method	Wavelength: 295nm; Improved selectivity; Suitable for mixture analysis	15
3	Levofloxacin	Simultaneous estimation method	Wavelength: 287–295nm; Solvent: Methanol; Used for combination drugs; Linearity: 5–25µg/mL	16

Table 3: Analytical methods development and validation for Levofloxacin by LC–MS/MS

Sr. No

Drug

Method

Description

References

Levofloxacin

LC-MS/MS method for plasma analysis

Column: C18 (50 × 2.1mm);

Mobile phase: Acetonitrile: 0.1% formic acid (70:30 v/v);

Flow rate: 0.3mL/min; Detection: ESI positive mode;

Run time: 2.5min;

Linearity: 1–5000ng/mL

Levofloxacin

Bioanalytical LC-MS/MS method

High sensitivity; Used for pharmacokinetic studies; Accurate quantification in biological matrices

Table 4: Analytical methods development and validation for Levofloxacin by HPTLC

Sr. No

Drug

Method

Description

References

Levofloxacin

HPTLC method for estimation in tablets

TLC plate: Silica gel 60 F254;

Mobile phase: Methanol: Ammonia (9:1 v/v);

Wavelength: 290nm;

Rf value: ~0.45

Levofloxacin

Stability indicating HPTLC method

Detection: UV at 287nm; Used for degradation studies; Suitable for routine QC

Table 5: Analytical methods development and validation for Levofloxacin combined dosage forms by UV–Visible Spectrophotometry

Sr. No	Drug	Method	Description	References
1	Levofloxacin, Ambrosoli	UV spectrophotometric method	Wavelength: Levofloxacin: 287nm; Ambrosoli: 244 nm; Solvent: Methanol; Linearity: 5–25µg/mL; Suitable for routine QC	22
2	Levofloxacin, Metronidazole	UV simultaneous estimation	Wavelength: Levofloxacin: 287 nm; Metronidazole: 277 nm; Solvent: Methanol; Linearity: 5–40µg/mL	24
3	Levofloxacin, Ofloxacin	Derivative spectrophotometric method	Wavelength: 290nm; High selectivity; Suitable for mixture analysis; Linearity: 2–20µg/mL	25

Table 6: Analytical methods development and validation for Levofloxacin combined dosage forms by RP-HPLC

Sr. No	Drug	Method	Description	References
1	Levofloxacin, Ornidazole	RP-HPLC method for simultaneous estimation	RP-HPLC; Wavelength: 294nm; Column: C18 (250 × 4.6mm, 5µm); Mobile phase: Acetonitrile: Phosphate buffer (65:35 v/v); Flow rate: 1.0mL/min; Retention time: Levofloxacin: ~3.2min; Linearity: 5–30µg/mL; Correlation coefficient: 0.999	21
2	Levofloxacin, Cefixime	Simultaneous RP-HPLC method	Wavelength: 290nm; Column: C18; Mobile phase: Methanol: Water (70:30 v/v); Flow rate: 1.0mL/min; Linearity: 10–50µg/mL; Good resolution of both drugs	23
3	Levofloxacin, Azithromycin	RP-HPLC method	Column: C18; Wavelength: 292nm; Mobile phase: Acetonitrile: Buffer (60:40 v/v); Flow rate: 1.2mL/min; Linearity: 5–25µg/mL	26

Table 7: Analytical methods development and validation for Levofloxacin combined dosage forms by HPTLC

Sr. No	Drug	Method	Description	References
1	Levofloxacin, Ornidazole	HPTLC method	TLC plate: Silica gel 60 F254; Mobile phase: Toluene: Methanol: Ammonia (7:3:0.5 v/v/v); Detection: 290nm; Rf: Levofloxacin ~0.42	27
2	Levofloxacin, Cefixime	HPTLC simultaneous estimation	Mobile phase: Ethyl acetate: Methanol: Water (8:2:1 v/v/v); Detection: UV at 287nm; Good separation achieved	28
3	Levofloxacin, Metronidazole	Stability indicating HPTLC	Detection: 288nm; Used for degradation studies; Suitable for QC	29

CONCLUSION:

Several methods have been reported in literature for the estimation of levofloxacin such as UV spectroscopy, HPLC, LC–MS/MS and HPTLC. Among these, HPLC and LC–MS/MS methods are most preferred due to their high sensitivity and accuracy. However, UV spectrophotometric methods are also useful for routine analysis as they are simple and cost-effective. Advances in various analytical tools will help in improving the drug quality control and their efficacy for therapeutic use.

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Received on 14.04.2026 Revised on 16.05.2026

Accepted on 11.06.2026 Published on 10.07.2026

Available online from July 25, 2026

Asian Journal of Pharmaceutical Analysis. 2026; 16(3):224-228.

DOI: 10.52711/2231-5675.2026.00034

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