INTRODUCTION:

Being an antimicrobial agent Levofloxacin hemihydrates are structurally related to nalidixic acid discovered in the 1960s. Being a fluoroquinolone antimicrobial used in use in urinary infections¹. Levofloxacin hemihydrates chemically called as (-)-(S)-9-fluoro2,3-dihydro-3-methyl-10-(4-methyl-1- piperazinyl) -7-oxo-7H-pyrido [1, 2, 3-de] - 1, 4 benzoxazine-6-carboxylic acid hemihydrate². It is the active S-isomer isolated from the racemic ofloxacin. It exhibited a broad spectrum of antibacterial activity against gram-positive and gram-negative bacteria³. It also exhibited potency against atypical pathogens like Mycoplasma, Chlamydia and Legionella⁴. The antimicrobial action is caused due to the inhibition of enzyme DNA gyrase (topoisomerase II and topoisomerase IV), essential for bacterial DNA replication, transcription, repair and recombination⁵.

The current literature reviles different spectroscopic and chromatographic methods for the development and validation of assay methods for the estimation of levofloxacin bulk drugs, pharmaceutical dosage forms and physiological fluids^6-14. In most of them, the use of different organic solvents was reported.

The present analysis aimed to develop and validate the precise, accurate and cost-effective analytical method for the estimation of Levofloxacine hemihydrates in bulk and tablet dosage forms. The methods were developed by derivative spectroscopic¹⁵ and RP-HPLC¹⁶ methods using the cheapest solvents. UV-Visible spectroscopic method for the estimation of levofloxacin using 0.1M NaOH and 0.1M HCl^17,18. Similarly RP-HPLC method was developed by Patel Dhara and Vaddeswarapu Madhavi using Glacial acetic acid: Methanol (45:55 % v/v)¹⁹ and Phosphate Buffer:ACN: Methanol (40:40:20v/v%)²⁰.

MATERIALS AND METHODS:

The analysis was carried out with a UV-visible spectrophotometer (Lab India 3000 spectrophotometer with UVWin5 Software v5.2.0.1104) and HPLC made up of CYBER LAB, software: UV-Vis detector. Digital weighing balance (Shimadzu AUX 220), p^Hmeter (INCO Lab) and ultra sonicator of (Citizen), Filtration membrane (0.22µ and 0.45µ) was used for analysis. Levofloxacin hemihydrates were obtained from Aurobindo Pharma Pvt. Lt yarrow chemicals in Mumbai, India. HPLC grade methanol, acetonitrile and triethylamine were from S.D. fine chemicals.

UV- Spectroscopic Method Development²¹:

Selection of solvent:

From the solubility data, water was chosen as the solvent for the analysis.

Preparation of standard stock solution of Levofloxacin hemihydrates:

Accurately taken 100mg of Levofloxacin hemihydrates and dissolved in 100ml of water. From the above solution taken 10.0ml of the solution to dilute to 100ml to obtain the concentration of 100µg/ml of Levofloxacin hemihydrates.

Preparation of working standard solution:

0.1, 0.2, 0.3, 0.4 and 0.5ml of solutions are taken from the above stock solution and diluted to 10ml with diluents to achieve a concentration range of 1-5μg/ml received and used for further investigation.

Determination of suitable wavelength:

The prepared 3μg/ml concentration of levofloxacin was scanned in UV visible spectrophotometer in the range of 200-400nm to get absorption maxima at zero, first and second-order derivative spectra.

Development of spectroscopic assay method:

Accurately weighed 20 tablets of 250mg equivalent weight Levofloxacin hemihydrates as per label claim marketed by IPCA Laboratories. An equivalent weight of 100mg was accurately powdered was taken and dissolved in 70ml of water in 100ml of the standard flask and sonicate for 30 minutes and filter by membrane filter and made up the volume made up to mark. Transferred 10ml from stock and diluted to 100 ml with solvent in 100ml volumetric flask to get a concentration of 100µg/ml. Again 0.4ml of the solution was withdrawn and diluted to 10ml and used for the assay Each solution was made in 3 sets and find out the absorbance of the above solution at 287, 296 and 282 nm, finally determine the concentration by direct comparison method using the formula.

C1 A1

------- = ------

C2 A2

Method Development and Optimization of Chromatographic Conditions²²

Preparation of mobile phase:

Mixed acetonitrile with orthophosphoric acid in methanol in a ratio of 50:20:30% v/v and degassed the mobile phase for 15 minutes.

Preparation of standard stock solution for RP-HPLC:

Weighed exactly and transferred 100mg of levofloxacin hemihydrate working standard to 100ml of clean, dry volumetric flasks, added approximately 100ml of diluent, and sonicated to completely dissolve and fill to the mark. From the above stock, the solution takes 10ml of solution and is further diluted to 100ml to get the concentration range of 100μg/ml.

Preparation of working standards for RP-HPLC:

From the above stock solution taken 0.2, 0.4, 0.6, 0.8 and 1ml of solution in 10ml of volumetric flask and made up the volume to get a concentration of 2-10 µg/ml.

Optimization of Chromatographic Conditions:

The chromatographic method was optimized with the mobile phase orthophosphoric acid: acetonitrile: methanol (50:20:30) at 287.36nm. The retention time was found to be 2.54 minutes.

Development of RP-HPLC assay method:

Accurately weighed 20 tablets of 250mg equivalent weight of Levofloxacin hemihydrates as per label claim marketed by Ipca Laboratories. A weight of 100mg accurately powdered was taken and dissolved in 70ml of diluents orthophosphoric acid: acetonitrile: methanol (50:20:30) in 100ml of the standard flask and sonicated for 30 minutes and filterd by membrane filter and made up the volume made up to mark. Transferred 10ml from stock and diluted to 100ml with solvent in 100ml volumetric flask to get a concentration of 100µg/ml. Again withdrawn 0.4ml of solution and diluted to 10ml and used for the assay. The solution was made in 3 sets and find out the retention time of the above solution, finally determine the concentration by a direct comparison method using the formula.

C1 A1

------- = ------

C2 A2

Validation Parameters:

The proposed method was extensively validated in terms of specificity, linearity, accuracy, precision, robustness, ruggedness, limits of detection (LOD) and quantification (LOQ) as per ICH guidelines^23,24. For all the parameters percentage of relative standard deviation was calculated.

Preparation of test solutions for a validation study of drugs:

Weigh and transfer 100mg levofloxacin hemihydrate as a standard into 100ml clean, dry volumetric flasks. Add approximately 70ml of solvent and mobile phase used during method development and sonicate to completely dissolve and make up to the mark with the same solvent (stock solution).From the stock solution prepared 1-5 µg/ml for derivative spectroscopic methods and 2-10 µg/ml for RP-HPLC methods. Each sample was separately prepared in 6 sets for each parameter.

Linearity:

A linearity study was carried out at 1-5 µg/ml for the derivative spectroscopic method and 2-10 µg/ml for RP-HPLC methods. Each sample was prepared in 6 sets and measured percentage relative standard deviation (%RSD).

Precision:

The method precision study was carried out at 2 µg/ml respectively.

Accuracy ( Percentage recovery studies):

The recovery study was carried out at 50,100 and 150 % levels for both methods. For the recovery studies, 3 dilutions were prepared using both standard drug and marketed formulation.

Ruggedness:

Analysis was carried out by different analysts to determine the ruggedness study. The ruggedness study was carried out at the concentration of 2 µg/ml respectively.

Robustness:

The robustness analysis was carried out under different conditions like changes in temperatures i.e., room temperature and 40°C also by the change in flow rate. For the robustness study concentration of 2 µg/ml was selected.

Detection Limit and Quantitation Limit:

The LOD and LOQ were determined by the standard deviation of the response and the slope of the calibration curve using the formula 3.3σ/S and 10σ/S criteria, respectively; where σ is the standard deviation of y-intercepts of regression lines and s is the slope of the calibration curve.

RESULT:

Figure-1: Zero-order (A), First order (B) and Second-order (C) derivative spectrum of Levofloxacin hemihydrate

Figure-2: Optimized chromatogram by RP-HPLC methods

Table-1: The Calibration data of Levofloxacin by derivative spectroscopic and RP-HPLC methods

S. no	Concentration (µg/ml)	Absorbance			Peak area
S. no	Concentration (µg/ml)	Zero-order (287nm)	First-order (296nm)	Second-order (282nm)	Peak area
1	1	0.1408	0.00031	-0.00060	-
2	2	0.2488	0.00060	-0.00040	26580
3	3	0.3871	0.00089	-0.00020	-
4	4	0.4988	0.00121	0.00001	55570
5	5	0.6303	0.00155	0.00020	-
6	6	-	-	-	83761
7	8	-	-	-	112824
8	10	-	-	-	141487

Figure-3: Calibration curve by zero order (A), first order (B), second order (C) and RP-HPLC method

Table-4: Summary of validation parameters by derivative and RP-HPLC methods

Sl. No	Validation Parameters*	Zero order	First order	Second order	RP-HPLC
1	Range (µg/ml)	1-5	1-5	1-5	2-10
2	λ_max	287nm	296nm	282nm	287 nm
3	Slope	0.124	0	0	14205
4	Intercept(c)	0.005	0	0	988.5
5	Correlation Coefficient(r²)	0.999	0.999	0.999	0.999
6	Assay at 4 µg/ml (%)	99.32 ± 1.21	98.32 ± 1.39	98.12 ± 1.56	99.47 ± 1.38
7	Percentage recovery at 100 % Level	98.26	100.9	100.43	100.23
8	Precision
A	Repeatability
	Morning (%RSD)	0.4202	1.733	1.92	1.19
	Evening (%RSD)	0.757	1.759	1.65	1.18
B	Reproducibility
	Day-1 (% RSD)	0.5652	1.499	1.561	1.29
	Day-2 (% RSD)	0.657	1.518	1.191	1.31
9	Ruggedness (% RSD)
	Analyst-1	1.13	1.064	1.93	1.13
	Analyst-2	1.15	1.109	1.87	1.22
10	Robustness (% RSD)
	Cool condition (8°C)	1.19	1.20	1.63	1.69
	Hot condition (50°C)	1.07	1.71	1.73	1.36
	Flow rate 0.8 ml/min	-	-		1.56
	Flow rate 1.5 ml/min				1.46
11	LOD (µg/ml)	0.05	0.03	0.02	0.9
12	LOQ (µg/ml)	0.15	0.09	0.06	2.7

*All the validation parameters were carried out in 6 spikes and the percentage RSD was calculated. Parameters 8, 9 and 10 were carried out at 2 µg/ml.

DISCUSSION:

The present work describes the development of a simple, precise and accurate derivative spectroscopic and RP-HPLC method for the estimation of levofloxacin hemihydrate in the tablet dosage form.

The spectroscopic method was developed by using water as a solvent. The absorption maxima were determined at 3 µg/ml. The solutions were scanned with a UV-Vis spectrophotometer (Lab India with UVWin5 software v5.2.0.1104 in the UV range 200-400 nm). Levofloxacin hemihydrate showed absorption maxima at 287.36 probe software of the instrument. The amplitudes of the corresponding troughs were measured at 287.2 and 296.2 nm (Figure 1). Levofloxacin hemihydrate follows linearity in the concentration range of 1-5 μg/ml in all methods. The method was further developed by the RP-HPLC method. The method was optimized by using Orthophosphoric acid: acetonitrile: methanol (50: 20: 30) as solvent at 287 nm. The retention time was 2.54 min with a tailing factor of less than 2 (Figure-2).

In all the methods, levofloxacin hemihydrate showing linear regression equations and Correlation Coefficient (r²) y = 0.124x + 0.005 (0.9999) zero order; and y = 0.000x-0.000006 (0.999) for first-order derivative method, y = 0.000x - 0.000 (0.999) in second order, y = 14205x - 988.5 (0.999) in RP-HPLC method (Table-1 and Figure-3). The assay of tablet dosage form was carried out at 4 µg/ml showing percentage purity of 99.32, 98.32, 98.12 and 99.47 % respectively at zero, first, second-order spectroscopy and RP-HPLC methods (Table-2).

The method has been validated according to the ICH guidelines. The recovery studies were carried out at three different levels; H. 50%, 100% and 150% levels by the zero, first and second-order spectroscopic method and the RP-HPLC method. The result showed a recovery of 95-101% by various methods (Table-3) The solvents used in this method for the analysis of levofloxacin hemihydrate in both bulk and formulation were very economical

The results showed that the method developed and validated in the current study is economical, accurate, precise and reproducible. The method can suitably be used for the determination of levofloxacin hemihydrate for the routine analysis and quantitative determination of levofloxacin in both active pharmaceutical ingredients and tablet formulations.

CONCLUSION:

The present analysis represents obtained is economical, accurate, precise and reproducible results. The method can be used for routine analysis and quantitative determination of levofloxacin in both active pharmaceutical ingredients and tablet formulations.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

All the authors would like to give thanks to management of School of Pharmacy, Guru Nanak Institutions Technical Campus, Hyderabad and Srikrupa Institute of Pharmaceutical Sciences, Siddipet for their kind support during the studies.

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Received on 22.12.2021 Modified on 09.06.2022

Research J. Pharm. and Tech 2023; 16(5):2239-2244.

DOI: 10.52711/0974-360X.2023.00368

Concentration (µg/ml)	Zero-order	First order	Second order	RP-HPLC
	Mean Percentage ± SD
4	99.32 ± 1.21	98.32 ± 1.39	98.12 ± 1.56	99.47 ± 1.38

Recovery Level	Zero-order	First order	Second order	RP-HPLC
Recovery Level	Mean Percentage recovery
50%	95.12	98.12	98.75	99.19
100%	98.26	100.9	100.43	100.23
150%	101.65	99.76	99.85	101.14