Validated Method for the Determination of Gemifloxacin in Bulk , Pharmaceutical Formulations and Human Serum by RP-HPLC : In Vitro Applications

Um método isocrático de cromatografia líquida de alta resolução de fase reversa (RP-HPLC) foi desenvolvido para determinação de gemifloxacin a granel, em formulações e soro humano a 270 nm. A separação cromatográfica foi adquirida em uma coluna Purospher STAR C18 (250 × 4,6 mm, 5 μm) usando a fase móvel metanol:água (90:10, v/v) ajustada para pH 2,8 com ácido fosfórico 85% em fluxo de 1,5 mL min a temperatura ambiente. As curvas de calibração mostraram-se lineares dentro do intervalo de 5-100 μg mL com um coeficiente de correlação de 0,9998. Os limites de detecção (LOD) e de quantificação (LOQ) foram 0,015 and 0,045 μg mL, respectivamente. Os resultados de precisão intrae inter-corridas e de exatidão foram 98,73-100,12%, que foram correlacionados através do teste t student. Este método foi aplicado para interações in vitro do gemifloxacino com elementos essencial e traço.


Introduction
Gemifloxacin (GMFX) is a fourth generation fluoroquinolone antibacterial compound with enhanced affinity for bacterial topoisomerase IV and is being used for the treatment of respiratory and urinary tract infections.2][3] It is particularly active against Gram-positive organisms including penicillin, macrolide, and quinolone resistant Streptococcus pneumoniae, 4 4-folds more potent than moxifloxacin against S. pneumoniae. 5Gemifloxacin has also shown potent activity against other major pathogens involved in respiratory tract infections, including Haemophilus influenzae and Moraxella catarrhalis and the atypical organisms, Legionella pneumophila, Chlamydia spp.and Mycoplasma spp. 6,7Furthermore, the compound has shown potent activity against many organisms that cause urinary tract infections and bronchitis. 8iterature survey revealed that few analytical methods have been reported for the estimation of GMFX, they include high performance liquid chromatography tandem mass, 9,10 microchip electrophoresis, 11 chiral highperformance liquid chromatography 12 and chiral countercurrent chromatography. 13,14Simple and sensitive ionpairing spectrophotometer methods have been described for the assay of gemifloxacin mesylate by Marothu et al. 15 Barbosa and co-workers 16 studied dissociation constants of series of compounds including diuretics and quinolones in several acetonitrile:water mixtures.
The aim of the present study was to establish an efficient, reliable, accurate, sensitive and reproducible

Chemicals and reagents
All chemicals and reagents were of analytical grade with purity percentage 99.89%.Gemifloxacin was a kind gift from Pharm Evo (Pvt) Ltd., Pakistan.Figure 1 shows the chemical structure of gemifloxacin mesylate.HPLC grade methanol was obtained from Merck Schuchardt OHG (Darmstadt, Germany).Each product was labeled and expiry dates were not earlier than two years, at the time of study.HPLC grade methanol were supplied by Tedia company, INC. (USA).
The essential and trace elements used were of analytical grade in the form of their hydrated salts as magnesium chloride hexahydrate (MgCl Concerning the pharmaceutical dosage form, Gemixa™ (Gemifloxacin 320 mg tablets by Bosch Pharmaceuticals (Pvt Ltd.)) was purchased from the local pharmacy.Expiry dates were not earlier than two years, at the time of study.

Statistical study
Standard regression curve analysis was performed by use of STATISTICA version 7.0 (USA), without forcing through zero.Linearity graphs were obtained by use of Microsoft Excel 2007 software.SPSS software version 10.0 (Carry, NC, USA) was used for the calculation of means, standard deviations, homoscedasticity of the calibration plots and Student's t-test.

Instrumentation
The UV-Vis spectrophotometer (Shimadzu 1601), integrated with a P IV computer loaded with UVPC version 3.91 was used to optimize the wavelength.The HPLC system consisted of an LC-20 AT VP Shimadzu pump and SPD-20AV VP Shimadzu UV-Vis detector, and the separation was achieved on a Purospher STAR C 18 (250 × 0.46 mm, 5 mm).The chromatographic and integrated data were recorded using a CBM-102 communication Bus Module Shimadzu.Sonicated by DGU-14 AM, and filtered through 0.45-micron membrane filter.Calibrated Pyrex glassware was used for the solution and mobile phase preparation

Standard preparation
Calibration standard solutions of GMFX were prepared by dissolving 1 mg mL -1 of drug using mobile phase as solvent and kept in 100 mL of volumetric flasks.Working solutions were prepared separately by making serial dilutions from the standard solution to obtain concentration between 5-100 µg mL -1 for GMFX.For quality control samples (QC), twenty tablets of gemifloxacin were powdered finely and an amount equivalent to 10 mg of GMFX was weighed and then dissolved in the mobile phase.Solutions with high, medium and low concentrations, i.e. 40, 50 and 60% were prepared, and then filtered through a 0.45 µm Millipore filter, in order to separate out the insoluble excepients by the same procedure as calibration standards but using different stock solutions.
All these solutions, calibration and QC were stored at 20 ºC.Once prepared, analyzed daily for inter and intraday variations of the method.20 µL of these solutions were injected into LC system and chromatographed

Procedure for human serum
Plasma sample, obtained from healthy volunteers, was collected and stored at −20 ˚C.To an aliquot of 1.0 mL plasma, 10 mL of acetonitrile was added and the mixture was vortexed for 1 min followed by centrifugation at 10,000 rpm during 10 min.It was then alienated supernatant by filtration (0.45 µm pore size membrane filter).An aliquot

Procedure for in vitro interaction studies
For interaction study stock solutions of GMFX and metals were prepared by dissolving 10 mg of the drug in 100 mL, using buffers of pH 1.0, 4.0, 7.4 and 9.0 as solvents, individually, then sonicated.Similarly stock solutions of metal salts ( were prepared in the same concentrations gemifloxacin solution and mixed with solution of metal in a flask.The final concentration of these solution was 50 µg mL -1 and the their flasks were then kept in water bath at 37 ± 5 o C for 3 h.Aliquots of 5 mL were withdrawn at an interval from 30 to 180 min, following filtration through normal filter paper then 0.45 mm filter paper to avoid any hindrance and then subjected to assay by RP-HPLC.Same procedure was repeated with heavy metals (lead (PbCO 3 ), arsenic (Ar 2 O 3 ) and silver (AgCl 3 )) for interaction study.

Method development
The aim of the present study was to develop a simple, isocratic, accurate and sensitive HPLC method for the determination of GMFX.Purospher STAR column (C 18 (250 × 0.46 mm, 5 mm)) provided the best peak shapes and efficiencies.The chromatographic conditions, especially the composition of the mobile phase, were optimized through several trials to achieve symmetric peak shapes for GMFX, as well as shorter run time.It was found that a mobile phase containing a high proportion of methanol gives symmetric peak shape.Therefore the final mobile phase was composed of methanol and water in ratio of 90:10 (v/v) providing good resolution.
In order to keep constant pH of the mobile phase, 85% phosphoric acid was added into mobile phase to achieve the desire pH.The mobile phase pH had a little impact on resolution and the best separations were observed at pH 2.8.The chromatographic conditions were optimized to achieve best separation and to get best resolution between analytes and to optimize chromatographic parameters like resolution, tailing factor and retention time.
Peaks were identified using retention times compared with those of standards.Retention time was 3.8 min for GMFX.For validation of analytical methods, the guidelines of the International Conference on the Harmonization of Technical Requirements for the Registration of Pharmaceuticals for Human Use 17 and USP 2002 18 were followed for the accuracy tests, precision, specificity, linearity, work strip and robustness of the method.

System suitability
It is an imperative module of method validation to make certain that the operational system is running appropriately throughout the analysis.The system was equilibrated with the initial mobile phase composition, followed by 10 injections of the same standard.These 10 consecutive injections were used to evaluate the system suitability on each day of method validation (Table 1).

Calibration curves
Calibration curves were characterized by different linear segments.These curves were obtained using the linear least squares regression procedure.These analysis results reveal good linear correlations (Table 1).The homoscedasticity of the calibration plots, tested by Friedman's tests, were found to be significantly linear over the tested ranges.

Recovery studies
The accuracy of the method was evaluated from the recovery results of spiked placebo samples.Appropriate portions of stock solution of drugs were spiked into blank placebo matrix to produce concentrations of 40, 50 and 60 µg mL -1 of the theoretical concentration.Mean recovery of spiked samples were in the ranges of 98.73-100.12%.Recovery tests were performed by adding known amounts of standard solutions to sample followed by analysis using proposed method.Three runs were performed for every concentration and then peak area was calculated (Table 2).[21][22] Precision Instrumental precision was determined by six replicate determinations of standard solution, i.e. repeatability.Method precision or intra-assay precision was performed by preparing six different samples involving different weightings.Each solution was injected in triplicate under the same conditions and the mean values of peak area responses for each solution were taken.0][21][22] All the results were correlated and found insignificant by student's t-tests indicating no remarkable difference in intra and inter day precision.

Specificity and selectivity
Figure 2 showed the typical chromatograms of drugs alone and with spiked plasma samples.No significant interference was observed from endogenous substances in drug free human plasma at the retention time of all drugs under study.

Ruggedness
The ruggedness of the method was established by determining GMFX, in bulk, dosage formulation and in human serum in two different laboratories (lab).Lab 1 was Research Institute of Pharmaceutical Sciences, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, while other lab was lab 9, Department of Chemistry, Faculty of Science, University of Karachi.For two different instruments at the same configuration, LC 20, on different days and different analytes (Table 2), the results showed to be in good limits.

Robustness
Robustness of the method was accomplished by designed modifications made to the method parameters, such as pH of the mobile phase, flow rate and composition.Therefore, five repeated samples were injected under small variations of each parameter.The method proved to be quite stable as there is no considerable drift in the factors as given in Table 3.

Application of the proposed method for in vitro interaction study
The interaction study between drugs and transition metals is an important research area in bioinorganic  chemistry.The action of many drugs is dependent on the coordination with metal ions, or/and the inhibition on the formation of metalloenzymes. 23The transition metals are integral part of an organic structure performing a vital function in the organism during the biological process of drug utilization in the body.The reduction of metals below certain limit results consistently in a reduction of physiologically important function. 24Although, the absorption of quinolone drugs is lowered when they are consumed simultaneously with multivitamins, magnesium or aluminium containing antacids and others bivalent cations.The proposed mechanism of the interaction is chelation between the 4-oxo and adjacent carboxyl group of quinolone and metal cations.Since these functional groups are required for antibacterial activity, it could be anticipated that all of the quinolones could be interacting with metal ions.Literature survey assembled a number of different complexation of quinolones.Efthimiadou et al. 25 discovered ciprofloxacin, cinoxacin, norfloxacin and nalidixic acid complexation with VO 2+ , Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ , Zn 2+ , MoO 2+ , Cd 2+ and UO 2+ , vanadyl complex with enrofloxacin and copper complexe with sparfloxacin. 26Skyrianou and co-workers 27 reported nickel complex with sparfloxacin.Psomas et al. 28 present ciprofloxacin interaction with Mn 2+ , Fe 3+ , Co 2+ , Ni 2+ and MoO 2 .Alkaysi and co-workers 29 published norfloxacin interaction with aluminum, magnesium and calcium.Iztok compiled interaction of 16 metals with eight quinolones. 30here have been reported complexes of ciprofloxacin with V(IV)O 2+ , Fe(III) 31 and two complexes of norfloxacin with Zn(II). 32n this paper, we also present application of our developed method by performing in vitro interaction of GMFX with different essential, trace elements and heavy metals.The interaction of moxifloxacin was carried out at 37 °C for 30 min at simulated gastric juice and buffers of pH 4.0, 7.4 and 9.0.Complex formation of gemifloxacin with metals was shown by a considerable decrease in AUC and a drift in retention time.

Interaction of GMFX with essential and trace metals
Almost all metals were interacting with gemifloxacin in simulated gastric juice and buffers of pH 4.0, 7.4 and 9.0.All the metals show good interaction with gemifloxacin at every pH.Availability of gemifloxacin with Mg, Mn, Fe and Cu was decreased to ± 38.74%, while with Ca, Co and Cd percentage decrease was found to be ± 46.31.Decrease in availability of gemifloxacin with Cr and Ni was ± 51.00%, whereas with Zn it was 26.81%.The % availability of gemifloxacin with Ca, Cr, Fe, Co, Mn, Ni and Cu was decreased to 61.92-47.06while with Zn and Cd ± 75.81 and Mg shows no significant interaction at pH 4.0.Availability of gemifloxacin with Co, Fe, Cr and Mn was up to 83.63-98.23%.While with Ni, Mg, Zn, Ca and Cu 50.11-66.76%at pH 7.4 and 9.0, availability of gemifloxacin with all metals was significant in the range of 53.8-61.52%.The results of interaction are shown in Table 4.

Interaction of GMFX with heavy metals
Almost every heavy metal interacts with GMFX at selected pH (Table 4).Percentage availability of GMFX with heavy metals at all pH was in between 43.36-85.84%which suggests significant interaction among gemifloxacin and heavy metals.

Conclusion
The proposed HPLC method is simple, isocratic, rapid, specific, accurate and precise for determination of GMFX in bulk, pharmaceutical dosage formulation and human serum has been developed for the first time.Hence, it can be recommended for the routine quality control and evaluation of clinical data of gemifloxacin.

Table 1 .
Regression and system suitability characters GMFX = gemifloxacin, S. E. E. = standard error of the estimate and S.E.= standard error.

Table 2 .
Accuracy and precision of gemifloxacin D1 = Intra-day, D2 = inter-day variations and df = degree of freedom.

Table 3 .
Robustness of the proposed method (n = 6) = retention time, K'= capacity factors, N = theoretical plates, T = tailing factor and R s = resolution. R

Table 4 .
Percentage recovery of gemifloxacin with essential and trace elements