Gemofloxacin (GEMI) is a potent antibiotic for the treatment of infection caused by gram-positive bacteria, especially streptococcus pneumonia. It is belonging to fluoroquinolone class of antibiotics and is the more active antibacterial agent among the agents in the same class. A reverse phase high performance liquid chromatography has been developed for the determination of GEMI in pharmaceutical dosage form. The separation was carried out using Hichrom packing Kromasil-100-C18 (250 mm×4.6 mm) with particle size of 5 μm and mobile phase consisting of acetonitrile: 10 mM phosphate buffer at pH 3 (15:85) at a flow rate of 1.0 mL/min. GEMI was monitored by fluorescence detector set at λex/λem 268 / 390 nm. The method was statistically validated with respect to linearity, limit of detection (LOD) and limit of quantification (LOQ), precision and accuracy. and the linearity. The method was found to be linear in concentration range of 10-150 ng/mL. The LOD and LOQ were measured to be 2.56 ng/ mL and 7.77 ng/mL, respectively. The proposed method was successfully applied for the determination of GEMI in tablets formulation.
Keywords: Gemifloxacin Mesylate; RP-HPLC; Fluorescence Detector; Pharmaceutical Formulations
Fluoroquinolones are gaining much interest since their inception in last decades of the previous century. They provide activity against both gram positive and gram-negative organisms by the virtue of their fluorine and piperazine moieties bearing little side effects [1,2]. GEMI is a light brown powder, slight soluble in water, chemically, it is 7-[(4Z)-3(aminomethyl)-4- methoxyimino-pyrrolidin-1-yl]-1-cyclopropyl-6-fluoro-4-oxo-1, 4dihydro-1, 8-napththyridine-3-carboxylic acid (Figure 1). It is fourth generation FQs antibacterial agent having affinity towards bacterial topoisomerase IV . GEMI was approved by the U.S. Food and Drug Administration for the treatment of the upper respiratory tract infections and demonstrate a broad-spectrum activity against many pathogenic grams negative and gram positive bacteria, including many of the so called atypical respiratory pathogens . A number of analytical methods have been reported for the determination of GEMI in pharmaceutical formulation and biological samples, including capillary electrophoresis [5,6], ion selective electrodes determination [7,8], spectrophotometric [9- 16], spectrofluorometric [9,10,15-21], HPLC with UV detector [22-32], diode-array detector  and HPLC with triple quadruple mass detector . HPLC method with Fluorescence detector (HPLC-FL) for the determination of GEMI in spiked human plasma was demonstrated by Al-Hadiya, et al. . In this method a mobile phase consisted mainly of acetonitrile was used, and the limit of detection (LOD) and limit of quantification (LOQ) were found to be 10 and 30 ng/mL, respectively. To best of our knowledge HPLC-FL for the determination of GEMI in pharmaceutical formulation have not been reported. Therefore, this work was devoted to developing an inexpensive, simple, fast, and sensitive HPLC-FL method for the quantification of GEMI in pharmaceutical formulation.
Chemical and Reagent
Gemifloxacin (GEMI) (98%) was obtained from Bayer (AG, Leverkusen Germany); Factive tablets (320 mg GEMI per tablet) LG life science Ltd, Kore lisansiyla Abdilbrahim ilacsan. VeTic.A.S. Maslak /Istanbul 0.3.052007, 210/86. Deionized water was used to prepare all solutions in this study.
High Performance Liquid Chromatography System (HPLC) Shimadzu Item LC-20AB, prominence Liquid pumps [Shimadzu- UFLC] DGU-20A5. Degasser prominence, CBM. 20A Communications Bus Module, Sil-20AC. Auto Sampler 20Mpa (max pressure) CTO- 20AC Column oven, Fluorescence detector RF-10AXL Shimadzu, LC, solution software. The analytical column used was Hichrom packing Kromasil-100-C18 (250 mm×4.6 mm) with particle size of 5μm. The mobile phase containing 10 mM sodium dihydrogen orthophosphate, adjusted to pH 3 with orthophosphoric acid and acetonitrile in the ratio 85:15. The flow rate was 1.0 mL/min; injection volume 20 μL, column oven temperature was 25°C. The fluorescence detections were carried out at λex/λem 268 / 390 nm.
Preparation of Standard Stock Solution of GEMI
100 μg/mL stock standard solution of the drug was prepared by dissolving 10 mg of GEMI in 1.0 mL of NaOH (0.1 M), transferred into a 100 mL volumetric flask and diluted to the mark with deionized water. 1.0 mL from the stock solution of GEMI (100 μg/ mL) was transferred into 100 mL volumetric flask and completed the volume with deionized water. The working solutions were made by dilute aliquot volume with the mobile phase.
Preparation of GEMI Sample Solution
10 tablets (Factive-320 mg of GEMI) were weighed, the average weight was determined, ground into a fine powder using mortar and mixed. An accurately weight of powder equivalent to 0.01g of GEMI was transferred into 100 mL volumetric flask, 1.0 mL of 0.01M sodium hydroxide was added and diluted to the mark with deionized water, to obtain 100 μg/mL of GEMI sample solution. The resulting solution was filtered through 0.22 μm nylon membrane filter and degassed by sonication.
Buffer solution of pH 3.0 was prepared from 10 mM H3PO4 and 10 mM Na2HPO4 and adjusted by a pH meter.
The developed method was validated in terms of linearity, limit of detection (LOD) and limit of quantification (LOQ), precisions, and recovery. In order to obtain these validation data, calibration curve were constructed based on peak area obtained at Six different concentrations (10 ng/mL, 30 ng/mL, 50 ng/mL, 70 ng/mL,100 ng/mL and 150 ng/mL) of GEMI standards. LOD is defined as the lowest amount of analyte that can be detected whereas LOQ is defined as the lowest amount of analyte that can be quantified. The LOD and LOQ were calculated based on the approach of ‘‘Standard Deviation of the Response and the Slope’’ whereby the
where K=3.3 for LOD and 10 for LOQ, SDa is the standard deviation of the intercept, and b is the slope of the calibration curve. Precision data was quantified by analyzing the peak areas of three replicates of three concentrations levels (10, 60, 150 ng/ mL)) within the calibration curves and calculating the RSD. For recovery study, GEMI tablet solution at concentration of 10 ng/mL was spiked with GEMI standard at three concentration level (10, 50, 140 ng/mL).
Results and Discussions
Several systematic trials were performed to optimize the chromatographic conditions for developing a sensitive, precise and accurate HPLC method for simple analysis for GEMI in pharmaceutical dosage forms. In the present method mobile phase mainly consisted of buffer pH 3 and acetonitrile [85:15 (v/v)], was found to be optimum. This mobile phase was found to be suitable sine the as chromatographic peaks obtained were free from tailing. The excitation and emission wavelength of λex/λem 268 /396, were used. Under the above conditions GEMI was eluted in 4.8 min that was shorter than the reported method (6.5 min) .
The calibration graph was generated using 20 μl injection loop. Six different concentrations of GEMI (10 ng/mL, 30 ng/mL, 50 ng/mL, 70 ng/mL,100 ng/mL and 150 ng/mL) were analyzed according to experimental conditions. Then the calibration curve was established according to the obtained response (peak area) and the concentrations of GEMI in standard solutions. The results show a good linear relationship. The calibration data was summarized in Table 1. The calibration curve and chromatogram of GEMI standard are shown in Figures 2 & 3, respectively.
Limits of Detection (LOD) and Limit of Quantification (LOQ)
LOD and LOQ were Determined Using the Formula:
where K=3.3 for LOD and 10 for LOQ, SDa is the standard deviation of the intercept, and b is the slope. The values of LOD and LOQ were found to be 2.5 and 7.77 ng/mL respectively. These values are better that the reported in the literature  that were found to be 10 and 30 ng /mL for LOD and LOQ, respectively.
Precision for the Determination GEMI
Precision was measured in terms of repeatability of application and measurement. Repeatability was assessed by injecting in triplicate standard of GEMI at three different concentrations within the linear range of the calibration curve, at 10, 60 and 150 ng/mL, Table 2.
Note: *values are mean of 3 determinations.
Recovery for Determination of GEMI
The recovery test was studied by spiking GEMI tablet solution (10 ng/mL) with standard GEMI at three concentration levels (10, 50, 140 ng/mL). The percentage recoveries for method ranged from 98.7 % to 100.6% were obtained as shown in Table 3. These values almost in agreement with reported one .
Note: *Recovery was calculated as the amount found/amount taken×100%
Values are means ± RSD for 3 determinations.
Analysis of Pharmaceutical Formulation
The proposed method was applied for the analysis GEMI in tablet formulation form. The method showed high accuracy for the determination of the studied drug. Chromatogram for GEMI tablet is shown in Figure 4. The proposed method has advantage of being virtually free from interferences by excipients, Table 4.
Note: (*values are means of 3 determinations).
A simple, accurate, precise HPLC method has been developed and validated for the quantification of GEMI in pharmaceutical formulation, according to the International Conference of Harmonization (ICH) guidelines for validation of analytical procedures [36,37]. The proposed HPLC methods have advantages over previously reported method  in terms of the simplicity, sensitivity, cost-effect. therefore, the method is practical and valuable for routine application in quality control laboratories for analysis of GEMI.
- Abdalla A Elbashir, Sara AM Ebraheem, Alawia HE Elwagee, Hassan Y Aboul-Enein (2013) New Spectrophotometric Methods for the Determination of Moxifloxacin in Pharmaceutical Formulations. Acta Chimica Slovenica 60(1): 159-165.
- Abdalla A Elbashir, Bahruddin Saad, Hassan Y Aboul-Enein (2010) Recent Developments of Enantioseparations for Fluoroquinolones Drugs Using Liquid Chromatography and Capillary Electrophoresis. Current Pharmaceutical Analysis 6(4): 246-255.
- BM Lomaestro (2000) Formulary 35: 961-968.
- (1996) Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. World Medical Association.
- Elbashir B Saad AA, Abd Salhin, MM Al-Azzam, H Aboul-Enein (2008) J Liq Chromatogr Related Technol 31: 1465-1477.
- E Kima, YM Koob, DS Chunga (2004) J Chromatogr A 1045: 119-124.
- Idress MO, Elbashir AA (2017) Current Trends in Analytical and Bioanlaytical Chemistry Development and Validation of Potentiometric ZnO Nanorods Modified Ion Selective Electrode for Determination of Gemi-floxacin in Pharmaceutical Formulation Curr Trends Anal Bioanal Chem 1: 50-56.
- Abo-talib NF (2013) Ion selective electrodes for stability-indicating determination of gemifloxacin mesylate. Anal Bioanal Electrochem 5(1): 74-86.
- Dalia Zidan, Omnia A Ismaiel, Wafaa S Hassan, Abdalla Shalaby (2016) Simple Spectrophotometric and Conductometric Methods for Determination of Gemifloxacin in Pure, Pharmaceutical Dosage Form and Human Urine. J Appl Pharm Sci 6(12): 136-143.
- El-Didamony AM, Abo-Elsoad MO (2015) Spectrofluorimetric and spectrophotometric methods for the determination of gemifloxacin in bulk and tablets. Main group chem 14: 59-70.
- Al-Tamimi SA (2013) Spectrophotometric and Spectrofluorimetric Methods for the Determination of Gemifloxacin Mesylate in its Pure and Dosage Forms using Eosin. Asian J Chem 25: 9272-9276.
- Radha SVVD, Apparao KM CH, Ramakrishina K (2012) Int JPharm 4: 618-621.
- Ebraheem SAM, Elbashir AA, Aboul-Eneinb H (2011) Spectrophotometric methods for the determination of gemifloxacin in pharmaceutical formulations. Acta Pharm sinica B 4: 248-253.
- S Dey, YV Reddy, B Krishna, SK Sahoo, PN Murthy, et al. (2010) Int J Anal Chem 1: 130-133.
- El Gammal RN, Hammouda MER, El-Wasseef DR, El-Ashry SM (2016) Int J Adv Chem 12: 5299-5306.
- Nuha Fathi Ali Dsugi, Abdalla A.Elbashir, Fakhr Eldin Osman Suliman (2015) Supramolecular interaction of gemifloxacin and hydroxyl propyl β-cyclodextrin spectroscopic characterization, molecular modeling and analytical application. Spectrochim Acta A Mol Biomol Spectrosc 151(1): 360-367
- Nagaraju P, Ravisankar P, Devala GCR, Rajendra YP (2014) Int J Chem Sci 12: 1382-1394.
- Al-Tamimi SA, Alarfaj NA, Aly FA, Al-Mohaimeed AM (2014) J Lumin 29: 127-131.
- Mahrouse, Hassan MA, Fawzy MG (2014) Acta Pharmaceut 64: 15-22.
- Atia NN, Mahmoud AM, El-Shabouri SR, El-Koussi WM (2013) Int J Anal Chem.
- Tekkeli SEK, Önal A (2011) J Fluoresc 21: 1001-1007.
- Mousavi SHH (2018) Braz J Pharm Sci 54 art. no. e17239.
- Durmus Z, Tekkeli SEK, Kiziltas MV, Onal A (2017) J chi chem soc 62: 3483-3489.
- Abdallah NA (2014) J Chromatogr.
- Kumar BA, De AK, B Pal (2014) Int J Pharm Tech Research 6: 1011-1017.
- Panchumarthy R, Garikapati D, Chapala D, Basha SKA, Nagabhairava SR, et al. (2013) J chem Pharm 6: 46-54.
- Begum MH, Agarwal NK, Duraivel S (2013) Int J Pharm Technol Biotechnol 5: 701-706.
- El-Bagary RI, Abo-Talib NF, Eldin MBN (2011) J chem Pharm 3: 562-570.
- Sultana N, Arayne MS, Shamim S, Naz A (2011) J Chin chem soc 58: 629-636.
- Sultana N, Arayne MS, Shamim S, Akhtar M, S Gul (2011) J Brazil chem soc 22: 987-992.
- Sugumaran M, Jotheeswari D (2011) Int J Pharm Sci Rev Res 6: 18-20.
- Mohammad Y, Pragat H, Harish A (2010) EJ Chem 7: 1621-1627.
- Sharif S, Khan I, Sheikh T, Sharif Y, Ashfaq M (2011) Acta Chromatogr 23: 95-107.
- Kadi AA, Angawi RF, Attwa MW, Darwish HW, Abdelhameed AS (2013) J Chem.
- Al-Hadiya BMH, Khady AA, Mostafa GAE (2010) Talanta 83: 110-116.
- (2005) Topic Q2 (R1). Validation of analytical procedures: Text and methodology. International Conference on Harmonisation (ICH).
- (1995) ICH-Q2A guideline for industry March 1995. Text on validation of analytical procedures.