Simultaneous determination of moxifloxacin and ofloxacin in physiological fluids using high performance liquid chromatography with ultraviolet detection

https://doi.org/10.1016/j.jchromb.2016.03.002Get rights and content

Highlights

  • Simultaneous determination of moxifloxacin and ofloxacin in physiological fluids using HPLC–UV detection is carried out for the first time.

  • This method is more sensitive compared to reported methods.

  • This method is applicable for analysis of both drugs in plasma, aqueous humor and pharmaceutical dosage forms.

  • Extraction is carried out using simple protein precipitation method.

Abstract

A novel, sensitive and validated RP-HPLC–UV method was developed for simultaneous determination of moxifloxacin and ofloxacin using timolol maleate as internal standard in physiological fluids. Different experimental parameters were optimized and validated according to international guidelines. Complete separation of the analytes was achieved with Kromasil 100-5C18 analytical column (250 mm × 4.6 mm × 5 μm), methanol and 0.05% trifloroacetic acid (TFA) (38:62 v/v) were used as mobile phase, pumped at flow rate of 1.1 ml/min in isocratic phase, column oven temperature maintained at 45 °C and detection wavelength of 290 nm. Protein precipitation method was applied to extract the drugs from human plasma and bovine aqueous humor samples using methanol as precipitating solvent. This method is linear in concentration range of 0.018–100 μg/ml for moxifloxacin and 0.014–20 μg/ml for ofloxacin. The recoveries of the method were 97.52 and 97.39% in human plasma for MX and OFN respectively, while in aqueous humor 94.48% for MX. The LOD values in plasma were found to be 10.0 and 8.00 ng/ml for MX and OFN respectively, while their respective LOQ values were 18.0 and 14 ng/ml. In aqueous humor the LOD and LOQ for MX were 16.0 and 24 ng/ml respectively. In future, this method will be used to study the pharmacokinetic profile of moxifloxacin and ofloxacin in biological fluids and pharmaceutical products.

Introduction

Moxifloxacin (MX), (Fig. 1), is fourth generation floroquinolone. Chemically, it is 1-cyclopropyl-6-fluoro-8-methoxy-7-[(4aS,7aS)-octahydro-6H-pyrrolo [3,4-b]pyridin-6-yl]-4-oxo-1,4-dihydro-3-quinolinecarboxylic acid [1]. MX is active against a wide range of microbes and has efficacy against both streptococci and staphylococci. It also has a reasonable activity against gram negative ocular pathogens [2]. MX has much better penetration into the ocular tissues than any other fluoroquinolones, the mean aqueous humor concentration of moxifloxacin (0.67 ± 0.50 μg/ml) is higher than besifloxacin (0.13 μg/ml ± 0.58) and gatifloxacin (0.13 ± 0.08 μg/ml) [3], [4]. MX is therefore frequently used to treat and prevent various bacterial keratitis, conjuctivitis and endophthalmitis [5].

Ofloxacin (OFN), (Fig. 1), is a broad spectrum second generation floroquinolone, prescribed to treat a number of bacterial infections. Chemically, OFN is (±)-9-fluoro-2,3-dihydro-3-methyl-10-(4-methyl-1-piperazinyl)-7-oxo-7H-pyrido[1,2,3-de]-1,4-benzoxazine-6-carboxylic acid [6]. Like other floroquinolones, ofloxacin is also efficacious against both Gram positive and Gram negative bacteria, and so approved for the treatment of gastrointestinal, respiratory and urinary tract infections [7]. OFN is also used to treat different ocular infections [8].

Fluoroquinolones attach to protein/DNA complex involving DNA gyrase and topoisomerase IV, thus interferes with the replication of DNA into daughter cells [9]. Various spectrophotometric [10], [11] and HPLC techniques [1], [12], [13], [14], [15], [16], [17] have been reported for the analysis of moxifloxacin alone and/or in combination with other drugs in various pharmaceutical products, human plasma and aqueous humor. Similarly various analytical methods are also available for the determination and estimation of ofloxacin in plasma, blood, pharmaceutical products and aqueous humor using spectrophotometric [18] and HPLC techniques [8], [19], [20], [21], [22], [23], [24], [25].

Simultaneous determination of MX and OFX in physiological fluids has been reported using HPLC coupled with fluorescent detector [26], [27] and UPLC–MS/MS detection [28], [29]. These equipments are generally expensive and also the complex techniques involved would add up to cost of the analysis. Using these expensive techniques for routine clinical/pharmaceutical analysis of these fluoroquinolones would be not cost-effective. In most of the developing countries, research laboratories, pharmaceutical industries and clinical settings generally use HPLC coupled with UV detector. It is thus required to develop a validated HPLC–UV method for the simultaneous determination of both these drugs in human plasma and aqueous humor samples. To our knowledge so far, no simultaneous method is reported for MX and OFX using HPLC–UV detection.

The objective of this proposed method is to analyze the aforementioned drugs simultaneously using timolol maleate as internal standard (IS). This HPLC–UV method is simple, fast, reliable, sensitive and cost-effective for the analysis of moxifloxacin and ofloxacin in physiological fluids and pharmaceutical products. This method is validated and optimized for various experimental conditions such as mobile phase composition, flow rate, internal standard, column oven temperature, sample size and detector wavelength [30]. This method will be used to study the pharmacokinetic profile of MX and OFN and for the analysis of both these drugs in pharmaceutical dosage forms. Along with this, we can also quantify MX in aqueous humor using this method.

Section snippets

Chemicals and reagents

Moxifloxacin (MX) (purity 99.9%) (Dr. Raza Pharma, Pvt., Ltd., Peshawar) and Ofloxacin (OFX) (purity 99.9%) (Saydon Pharmaceutical Industries Pvt., Ltd., Peshawar) and internal standerd Timolol maleate (purity 97.5%) (Schazo Pharma. Pvt., Ltd., Lahore), were kindly supplied by these local pharmaceuticals. HPLC grade methanol, acetonitrile, dichloromethane, ethanol and triethylamine were acquired from Sigma–Aldrich (Oslo, Norway) while trifloroacetic acid (TFA) and diethyl ether were purchased

Results and discussions

The proposed method is accurate, reliable, economical and novel because simultaneous determination of ofloxacin and moxifloxacin in plasma was carried out for the first time using HPLC/UV method. The method is optimized and validated for analysis of ofloxacin and moxifloxacin in biological and pharmaceutical samples according to the standard guidelines [31], [32]. Moxifloxacin was appropriately separated applying this method in standard mixtures, spiked plasma and aqueous humor samples as shown

Conclusion

The developed RP-HPLC–UV method is new, sensitive, fast and cost effective. This method is validated in accordance with ICH guidelines and optimized for different experimental parameters for simultaneous determination of moxifloxacin and ofloxacin in biological fluids and pharmaceutical dosage forms. The analytical conditions and the mobile phase used provided good separation of the analytes in less than 9 min. The developed method offers the advantage as moxifloxacin and ofloxacin are

Acknowledgements

The author is extremely grateful to the University of Peshawar for providing research facilities and especially thankful to Dr. Zafar Iqbal (Meritorious Professor) for his kind guidance.

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