Quantitative Determination of Levofloxacin in Ophthalmic Solution by High- Performance Liquid Chromatography

doi:10.21203/rs.3.rs-3411084/v1

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Quantitative Determination of Levofloxacin in Ophthalmic Solution by High- Performance Liquid Chromatography

https://doi.org/10.21203/rs.3.rs-3411084/v1

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Levofloxacin, as a prototypical of the third generation of fluoroquinolones, is an antimicrobial agent routinely administrated for treating bacterial keratitis. Levofloxacin is available under different trade names as liquid pharmaceutical formulations, such as infusions and eye drops. This paper reports a fast, simple, accurate and precise HPLC technique for levofloxacin determination in liquid pharmaceutical formulations. The HPLC method was applied with Photodiode Array Detectors, and measurements were conducted at 294 nm UV-Vis wavelength. The technique was developed to immediately estimate levofloxacin in the Oftaquix 5 mg/mL ophthalmic formulation. The ODS-phenyl column was used and maintained at 30 ± 2 °C and 294 nm λmax conditions. The mixture of acetonitrile:0.1% trifluoroacetic acid (18:82 v/v) was used as a mobile phase with a flow rate of 1.5 ml/min. Levofloxacin peak was eluted at 9.5 min with good resolution. The method was validated in relation to system suitability, accuracy, linearity, and precision in agreement with ICH and FDA guidelines. The results indicated that the established HPLC-DAD method in this study is simple and accurate and can be applied for routine analysis of levofloxacin amounts in pharmaceutical formulations and bioequivalence studies in quality control departments and the pharmaceutical industry.

Levofloxacin

HPLC

eye drops

validation

ophthalmic

drug analysis

Infectious disease by microbial infections is one of the major catastrophes in medical and biomedical fields, which impose considerable economic, financial, and health-related burdens on patients and healthcare systems. For example, bacterial keratitis is a bacterial infection of the cornea and may lead to vision loss or blindness[1]. The cornea infection by Pseudomonas aeruginosa may arise following extended contact lens wear, eye surgery, and other pathogenic-involving situations. The treatment options may depend on microorganism culture and sensitivity tests by prescribing proper antibiotics[2]. Levofloxacin is a prototypical of the third generation of fluoroquinolones, exerting broad-spectrum antimicrobial agent against both Gram-positive and Gram-negative bacteria [3]. Besides bacterial keratitis, levofloxacin could also be successfully prescribed to treat other infectious diseases, such as chronic bacterial prostatitis, lower respiratory tract infections, urinary tract infections and H. pylori infections. Chemically, levofloxacin is a chiral fluorinated carboxyquinolone, which is the (-)-(S)-enantiomer of the racemic drug substance ofloxacin (Fig. 1)[4]. It is an amphoteric molecule (pKa1 and pKa2 values of 6.22 and 7.81, respectively) with a log P of 0.59 at the isoelectric pH of 7.10. Levofloxacin topical ophthalmic solution (5 mg/mL) is indicated for treating corneal ulcers and bacterial ocular infections.

The antibacterial activity of levofloxacin is related to inhibiting the enzymes (i.e., topo-isomerase II, topo-isomerase IV, and gyrase) responsible for separating bacterial DNA. It blocks cell transcription, replication, repair and recombination and, consequently, results in cell death[5–8]. According to the reported structure-activity data, the fluorine atom at position 6 of the naphthyridine ring helps the broaden levofloxacin activity spectrum against both Gram-negative and Gram-positive pathogens (Fig. 1)[3, 4, 9]. It is shown that the antibacterial activity of the S isomer of levofloxacin is approximately 130 times higher than the R- isomer[3].

The bacterial killing activity of quinolones shows a concentration-dependent manner and increases by increasing the drug concentration. In this regard, as the serum drug concentration of levofloxacin rises to around 30 times of the minimum inhibitory concentration (MIC), the antiseptic and bactericidal activity increases [5, 9, 10]. However, the topical administration of levofloxacin drops in treating bacterial keratitis exhibits lower bioavailability due to the eye’s unique anatomical and physiological constrictions. Therefore, the bactericidal effect of levofloxacin in treating bacterial keratitis is provided with a repeatable dose in defined time intervals. Although the repeated administration of the drug decreases the patient’s compliance, it also prevents the resistance of the bacteria to the treatment[2, 10]. The other factor that may influence the effectiveness of the treatment is the stability of the substance in pharmaceutical formulations, which impacts the effective concentration of the drug. As a result, quantitative determination of drug concentration in pharmaceutical formulations and also in human plasm is an essential parameter to consider in levofloxacin administration to obtain better clinical outcomes.

Until today, several analytical methods have been introduced to quantify levofloxacin amount in different dosage forms like ¹H NMR spectroscopy, ultra-performance liquid chromatography (UPLC), capillary electrophoreses, and High-performance liquid chromatography coupled with Ultraviolet (HPLC-UV), fluorescence (HPLC-FL), and tandem mass spectrometry (HPLC-MS/MS),) detectors[11]. In the HPLC methods, different kinds of additives were employed as mobile phase components for improving peak shape and resolution. For example, Triethanolamine (TEA), tetrabutylammonium bromide (TBAB), butadiene styrene brominated ammonium (BSBA), tetrabutylammonium hydrogen sulfate (TBHS), sodium or potassium phosphate buffers, etc. have been used in the mobile phase to improve peak shape[2, 11–13]. The other methods were also conducted under gradient elution method using formic acid, trifluoracetic acid, and methanol at minuscule concentrations (0.05–0.1%) as mobile phase components[14, 15]. However, these methods result in a relatively long separation time (about 13 min). On the other hand, using additives in the mobile phase in gradient elution exerts additional disadvantages, including slow equilibrium time, reduced column lifetime, tailing peak, early elution, late elution, and artefact peaks[16, 17]. Moreover, many of these HPLC methods are based on specific detectors that are not widely available in all laboratories because of the high cost of the equipment. In addition, although different HPLC methods have been applied to determine and quantify levofloxacin and its various derivatives in human plasma and standard solutions, the application of the HPLC method for determining levofloxacin concentration in real and pharmaceutical solutions has not been reported in previous literature.

With this background in mind, this study aimed to develop and validate a rapid, simple, economical, and precise HPLC method for quantitatively determining levofloxacin in ophthalmic and other pharmaceutical solutions.

Material and reagents

Levofloxacin standard was kindly gifted from Pioneer Pharmaceutical Industry, Iraq. All solvents used in this study were HPLC grade, and all reagents were analytical grade. Acetonitrile and trifluoroacetic acid were purchased from Merck, Germany. Water was purified with Milli-Q® Plus, Millipore System. Membrane filters with a pore size of 0.45 µm were acquired from Pall Life Sciences, India and all solvents and solutions were filtered through membrane filter and degassed before use.

Instrumentation

HPLC system: High-pressure liquid chromatograph model waters alliance 2695 HPLC system (Waters Corporation, Milford, MA, USA) equipped with autosampler injection system, quaternary pump, vacuum degasser, Photodiode Array Detector (DAD) with waters 2487 dual absorbance UV/Vis detector. The 2487 UV-Vis wavelength provides the most sensitive and versatile absorbance detection on HPLC.

Column: The analytical column was a phenyl L11 stationary column (250*4.6mm, 5µm, GL Sciences Inc. JP).

Sample Preparation

Stock solution (1000 µg/mL of levofloxacin) was prepared by transferring an appropriate volume of Oftaquix 5 mg/mL ophthalmic solution (Santen pharmaceutical industry, Japan) into a volumetric flask and diluted with diluent (acetonitrile:water 18:82 v/v) to volume 100 mL, shake well and then sonicated for about 10 min. The solution was then filtered through 0.45 µm pore size, and 20 µL of filtered solution was injected into the HPLC system. All solutions were prepared fresh each day.

Method evaluation and HPLC condition

The method was optimized under different experimental conditions to evaluate the optimum mobile phase ratio, column temperature, flow rate, and column stationary phase type. The mobile phase consisted of acetonitrile:0.1% trifluoroacetic acid (18:82 v/v). The injection volume was 20 µL, and all solutions, including the mobile phase, were sonicated 10 min before use. The UV detection was made at 294 nm for all experiments. Best separation was obtained by isocratic elution at a 1.5 mL/min flow rate. All analyses were done at constant temperature (30 ± 2°C).

Preparation of Calibration standard

Stock solution with a concentration of 1000 µg/mL was prepared by transferring an accurate amount of levofloxacin (from Oftaquix 5 mg/mL) and dissolving in diluent consisting of acetonitrile:water (18:82 v/v). Series solutions with five different concentration levels (50.19, 75.28, 100.38, 125.47, and 150.57 µg/mL) were prepared from the standard stock solution and conveniently diluted with the diluent solution. Each concentration was injected five times, and mean values of peak areas were plotted against concentrations.

Validation parameters

Validation parameters such as linearity, the limit of detection (LOD), limit of quantification (LOQ), accuracy, specificity and precision were carried out to determine the accuracy and precision of the obtained method according to the ICH (International Conference on Harmonization) guidelines. The sensitivity of measurements was estimated in terms of LOD and LOQ using the following equations[18]:

LOD = 3.3 × N/P
LOQ = 10 × N/P

Where N is the standard deviation of the peak areas of the levofloxacin (n = 5) and P is the slope of the corresponding calibration curve.

The establishment of a rapid, straightforward and precise method for determining the drug in pharmaceutical formulations and human plasma at the pharmacokinetic range is essential for drug analysis and bioequivalence studies in many laboratories. To this end, the best resolution of peak shapes at the chromatographic conditions was examined. Parameters like the symmetry of peaks, capacity factor and theoretical plate were studied to select the best mobile phase. It was observed that the mobile phase containing acetonitrile: 0.1% trifluoroacetic acid (18:82 V/V) in a flow rate of 1.5 mL/min at the optimum wavelength of 294 nm gives the best isolation of peak response of levofloxacin in both standard and sample solution with no detection in placebo solution as it is shown in the respective chromatograms in Figs. 2, 3 and 4 respectively. The retention time of levofloxacin at the flow rate of 1.5 mL/min is obtained at 9.5 min, which characterizes a short analysis time.

System suitability test

The system suitability test was performed before the validation step according to the FDA guidelines to ensure the instrument was performing well. In this regard, the established HPLC method passed all the required criteria in terms of plate count (> 2000), tailing factor (< 2.0) and %RSD value (< 1.0%). Therefore, the developed method was proved to give a good performance in the quantification of levofloxacin in standard and sample solutions.

Method Validation

The established method was validated to ensure accuracy, linearity, and recovery as required by ICH guidelines.

Linearity of Response

Different concentrations of the standard levofloxacin solution ranging from 50 µg/mL to 150 µg/mL were analyzed to obtain the linear calibration curve. Each concentration was injected five times, and an average peak area was plotted versus the respective concentration. The result showed an excellent correlation between concentration and peak area with a correlation coefficient (R²) of 0.9999. The calibration curve of levofloxacin is shown in Fig. 5, and the data used to establish the standard calibration curve are also summarized in Table 1.

Table 1

The data used to establish the standard calibration curve of levofloxacin.
Level	Concentration (µg/mL)	Area	Average area
50%	50.19	3202975.615	3517782.715
		3599406.283
		3596082.301
		3599022.267
		3591427.11
75%	75.28	5440183.623	5435350.571
		5437422.66
		5435823.793
		5429380.419
		5433942.36
100%	100.38	7226640.59	7214718.931
		7209162.866
		7215224.796
		7208150.723
		7214415.678
125%	125.47	9049063.412	9046726.799
		9041729.719
		9056174.365
		9032871.557
		9053794.941
150%	150.57	10881339.98	10881317.23
		10870525.53
		10878365.22
		10891160.02
		10885195.39

The equation of the calibration curve obtained was y = 73077 x − 116198. The correlation coefficient was 0.9999 (Fig. 5 and Table 2). The present method showed a good linear range with respect to the methods reported in previous studies[19].

Table 2

Results of validation of the HPLC-DAD determination of levofloxacin in Oftaquix 5 mg/mL ophthalmic solution.
Parameters	Results
Range (µg/ml)	50 to 150
Slope	73077
Intercept	-116198
Determination coefficient (R²)	0.9999
LOD (µg/ml)	2.13
LOQ (µg/ml)	6.47

Precision

The precision of any analytical procedure gives evidence of unsystematic errors. It is also a status agreement between sequences of measurements obtained from numerous samplings of the identical homogeneous sample solution under defined conditions. The precision was carried out at two levels: interday and intraday. Intraday was completed by examining the intermediate concentration of the drug, while interday was measured by evaluating over consecutive days for the same drug concentration six times. The percentage of relative standard deviation (%RSD) value was calculated. The data obtained from interday and intraday %RSD is less than 2% for levofloxacin, confirming that the established method is precise. The results of inter and intraday precision analysis are summarized in Table 3.

Accuracy

Recovery was examined by adding a known amount of levofloxacin standard corresponding to 75, 100, and 125% of the labelled claim of the drug. At each level, three readings were obtained (n = 3). The percentage of recovery of the drug ranged from 99.4–101.2%. The result is shown in Table 4, which indicates excellent accuracy was made at each added concentration.

Table 3

Result for inter and intraday precision.
Precision
	Day 1	Day 2
	Analyst 1	Analyst 1
	95.730%	97.33%
	95.729%	97.16%
	97.318%	96.47%
	97.341%	97.00%
	96.003%	97.36%
	95.936%	97.29%
Mean	96.465%	97.06%
SD	0.007951775	0.003364746
RSD%	0.824%	0.347%

Table 4

%RSD and accuracy values.
Standard Data
Weight	dil. Factor	Purity as is	Conc. µg/mL	Area		Average Area		SD	RSD%
			100.379	7209163.00000		7214719.2000		7356.79	0.1%
				7208151.00000
				7214416.00000
				7215225.00000
				7226641.00000
Accuracy - recovery of levofloxacin
Level	Amount spiked µg/mL	Area			Amount recovered µg/mL	%Recovery		Average Recovery	%RSD
75%	75.28		5467342		76.07	101.04%		101.03%	0.08%
			5462085		75.99	100.94%
			5470237		76.11	101.09%
100%	100.38		7172160		99.79	99.41%		99.38%	0.04%
			7166670		99.71	99.33%
			7172160		99.79	99.41%
125%	125.47		9109131		126.74	101.01%		101.18%	0.19%
			9121820		126.91	101.15%
			9144065		127.22	101.39%
Average							100.53%

RSD and SD denote relative standard deviation and standard deviation.

The percentage recovery of the spiked placebos should be 100 ± 2.0% for the average of each set of three weights. Each individual, sample recovery should lie within the range of 98–102%.

Stability in Solution and in the mobile phase

The assay of levofloxacin in solution stability confirmed that API was stable in diluent solution at ambient temperature (24 ± 2°C) for up to 48 h with %RSD less than 1.0%.

Eye drop application

The proposed method has been applied for levofloxacin analysis in Oftaquix 5 mg/mL ophthalmic solution. The drug content in each sample was calculated by comparing it with the standard solution. The amount of 4.96 mg/mL was found in commercially available eye drops (Table 5). The mean assay for six samples was 99.3%, with an %RSD value of 0.84% for six samples.

Table 5

Percentage of RSD value.
Drug	Label claim	Amount found	Assay %	RSD %
Levofloxacin	5 mg/mL	4.96 mg/mL	99.3%	0.84%

A new, rapid, and simple HPLC-DAD method was optimized and validated according to the ICH and FDA guidelines in terms of linearity, LOD, LOQ, accuracy, precision, and stability. A good system performance was observed for the established method based on FDA requirements. The developed method showed a good linear relationship between levofloxacin concentration versus area at 50–150 µg/mL ranges. The correlation coefficient was 0.9999. The result of the validation of the HPLC method of levofloxacin eye drops indicated that the method is accurate, linear, and precise, and samples are stable during performing analysis and up to 48 h. In general, the established HPLC-DAD method in this study is simple and accurate and can be applied for routine analysis of levofloxacin amounts in pharmaceutical formulations and bioequivalence studies in quality control departments and the pharmaceutical industry.

Ethical Compliance

Not Applicable

Conflict of Interest Statement

There are no conflicts to declare.

Data availability

All data generated or analyzed during this study are included in this published article.

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No competing interests reported.

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Quantitative Determination of Levofloxacin in Ophthalmic Solution by High- Performance Liquid Chromatography

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Version 1

Abstract

Figures

Introduction

Experimental

Material and reagents

Instrumentation

Sample Preparation

Method evaluation and HPLC condition

Preparation of Calibration standard

Validation parameters

Results and discussion

System suitability test

Method Validation

Linearity of Response

Precision

Accuracy

Stability in Solution and in the mobile phase

Eye drop application

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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