INTRODUCTION:

Gatifloxacin sesquihydrate (GS) (Fig. 1) is a synthetic broad-spectrum 8-methoxyflouroquinolone antibacterial agent for oral or i.v. administration. Chemically, GS is (±)-1-cyclopropyl-6-fluoro-1,4-diydro-8-methoxy-7-(3-methyl-1-piperazinyl)-4-oxo-3-quinoline carboxylic acid sesquihydrate.¹ GS is active against both gram-negative and gram-positive bacteria. It is used in the treatment of susceptible infections, including respiratory and urinary tract infections.² The absolute bioavailability of gatifloxacin is 96 % and peak plasma concentration is 3.4 ± 0.6 µg/ml, usually occurring 1-2 hours following the 400 mg of oral dose.³ Various methods have been reported in literature for the analysis of GS. Fluorimetric,⁴ HPLC,^5-6 reverse-phase HPLC/electrospray ionization mass spectrometry,⁷ liquid chromatography/electrospray tandem mass spectrometry⁸ and HPTLC^9-10 have been reported for quantitative determination of gatifloxacin. Spectrofluorimetric^11-12 and HPLC^13-15 for determination of gatifloxacin in biological fluids have also been reported.

However, to our knowledge, no article related to the stability-indicating HPLC determination of gatifloxacin from bulk drug and pharmaceutical formulations has ever been mentioned in literature. According to the international conference in harmonization guidelines [ICH Q1A (R2)] entitled “stability testing of new drug substances and products”, stress testing of drug should be carried out to elucidate the inherent stability characteristics of the active substance.¹⁶

The aim of the present work was to develop an accurate, specific, precise and stability-indicating HPLC method for determination of gatifloxacin sesquihydrate (GS) in the presence of its degradation products from a bulk drug and different pharmaceutical formulations and its application in quantitative determination of GS in human plasma.

EXPERIMENTAL:

Chemicals and Reagents

GS standard powder was kindly gifted by Alembic Ltd., Vadodara, India. Tablet, eyedrop and infusion were purchased from local pharmacy. All formulations were manufactured by Cipla Ltd.

Acetonitrile and methanol (HPLC grade) were purchased from Qualigens Fine Chemicals, Mumbai, India. Anhydrous sodium acetate, hydrochloric acid, sodium hydroxide pellets, hydrogen peroxide, triethylamine and o-phosphoric acid were also purchased from Qualigens Fine Chemicals, Mumbai, India and were of analytical grade.

Water for the HPLC analysis was generated by reverse osmosis using Milli-Q water system (Millipore Co., Bedford, MA, USA)

Figure 1 Structure of gatifloxacin sesquihydrate.

HPLC Instrumentation and Conditions

Chromatography was performed on Shimadzu (Shimadzu Corporation, Kyoto, Japan) chromatographic system equipped with Shimadzu LC-20AT pump and Shimadzu SPD-20AV absorbance detector. Samples were injected through a Rheodyne 7725 injector valve with fixed loop at 20 μl.

Table 1.Linear regression data for calibration curve (n=3)

Parameters
Linearity range	0.5-100 µg/ml
Correlation coefficient (r²)	0.9999
Slope ± S.D.	75.169 ± 0.005
S.E. of slope	0.003
Intercept ± S.D.	0.114 ± 0.10
S.E. of intercept	0.057

Table 2.Intra-day and Inter-day Precision

Actual Concentration (µg/ml)	^aIntraday measured concentration Mean (µg/ml) ± S.D., % RSD	^bInterday measured concentration Mean (µg/ml)±S.D., %RSD
0.5	0.497 ± 0.005, 1.10	0.496 ± 0.008, 1.58
45	45.008 ± 0.062, 0.13	45.082 ± 0.190, 0.42
100	99.954 ± 0.104, 0.10	99.997 ± 0.046, 0.04

^a mean concentration of six determinations.

^bmean concentration of nine determinations.

The chromatographic separation was performed using a μBondapackTM ODS C₁₈ (300 mm X 3.9 mm i.d., 10 μm particle size) column (Waters, Milford, Massachusetts, USA).

Separation was achieved using a mobile phase consisting of acetonitrile and sodium acetate buffer pH 3.4 (0.2 % triethylamine was added in buffer and pH of buffer was adjusted to 3.0 with o-phosphoric acid) in the ratio of 25:75 v/v, pumped at a flow rate of 0.8 ml/min. The eluent was monitored using UV detector at a wavelength of 293 nm. The column was maintained at ambient temperature and an injection volume of 20 μl was used.

The mobile phase was vacuum filtered through 0.45 μm nylon membrane filter followed by degassing in an ultrasonic bath (Analytik instruments, Mumbai) prior to use.

Data acquisition and integration was performed using Spinchrome software (Spincho biotech, Vadodara).

Preparation of Sodium Acetate Buffer pH 3.4:

50 ml of 0.1 M sodium acetate was mixed with 950 ml of 0.1 M acetic acid. pH was adjusted with acetic acid if necessary.

Preparation of Working Stock Solution:

Stock solution of gatifloxacin was prepared by dissolving 10.7 mg GS (equivalent to 10 mg gatifloxacin) in 10 ml methanol. The solution was shaken completely by hand and sonicated for 2 min in ultrasonic bath. 5 ml of the filtered solution was transferred to the 50 ml volumetric flask and diluted up to the mark with mobile phase to produce a working stock solution of 100 µg/ml.

Calibration Curve of Gatifloxacin:

Aliquot ranging from 0.5 ml to 8.5 ml were taken, from working stock solution, in 10 ml volumetric flask and diluted to 10 ml with mobile phase to give final concentration of 0.5, 1, 5, 25, 45, 65, 85 µg/ml. Injections of 20 μl were made for each concentration and chromatographed under the condition described in section 2.2. Calibration graph was constructed by plotting peak area versus concentration of gatifloxacin and the regression equation was calculated.

Table 3.Recovery Study

Excess drug added to analyte (%)	Theoretical Content (µg/ml)	^a Amount Found (µg/ml)	Recovery (%) ± S.D. , %RSD
0	25	24.99	99.99 ± 0.001, 0.01
80	45	46.09	102.40 ± 0.405, 0.87
100	50	50.18	100.37 ± 0.629, 1.25
120	55	55.49	100.90 ± 0.582, 1.05

^a mean concentration of six determinations.

Table 4.Stability in sample solution by HPLC method for GS

Actual conc. (µg/ml)	^a Measured conc. (µg/ml)	S.D.	% R.S.D.
0.5	0.49	0.006	1.23
45	44.65	0.572	1.28
100	99.82	0.247	0.24

^a mean concentration of three determinations.

Method Validation:

The method was validated with respect to parameters including linearity, limit of quantitation (LOQ), limit of detection (LOD), precision, specificity, recovery and robustness.¹⁷

Linearity:

Linearity of the method was studied by injecting eight concentrations of drug prepared in the mobile phase in the range of 0.5-100 μg/ml in triplicate in to the HPLC system and keeping the injection volume constant (20 μl).

LOD and LOQ:

LOD and LOQ were separately determined at a signal-to-noise ratio (S/N) of 3 and 10. LOD and LOQ were experimentally verified by diluting known concentration of gatifloxacin until the average responses were approximately 3 or 10 times the standard deviation of the response for six replicate determinations.

Precision:

For system precision, repeatability of peak areas were carried out using six replicates of the same concentration (100 μg/ml) and was expressed in terms of percent relative standard deviation (%R.S.D.). For method precision six injection of three different concentrations (0.5, 45, 100 μg/ml) were given on the same day for intra-day precision. Three injections of three different concentrations (0.5, 45, 100 μg/ml) were given different day for inter-day precision. The intra- and inter-day variation was expressed in terms of standard deviation (S.D.) and % relative standard deviation (% R.S.D.).

Specificity:

The specificity of method was established through study of resolution factors of the drug peak from the nearest resolving peak and also among all other peak.

Recovery Study:

Recovery study was carried out by applying the method to drug sample to which known amount of gatifloxacin corresponding to 80, 100, 120% of label claim had been added (standard addition method). At each level of the amount six determinations were performed. This was done to check recovery of the drug at different levels in the formulation.

Robustness:

To evaluate LC method robustness, a few parameters were deliberately varied. The parameters included variation of flow rate, percentage of acetonitrile in the mobile phase and acetonitrile of different lots. Robustness of the method was done at three different concentration levels 0.5, 45 and 100 μg/ml.

Preparation of Sample Solutions for Assay:

Assay of Tablet:

To determine the gatifloxacin in tablet (label claim: 400 mg/tab), twenty tablets were accurately weighed and finely powdered. An accurately weighed amount equivalent to 10 mg of gatifloxacin was transferred in to 10 ml volumetric flask, dissolved in 8 ml of methanol and volume was made up to the mark with the same solvent. The volumetric flask was sonicated for 2 min to effect complete dissolution and then filtered. The concentration of gatifloxacin in sample stock solution was 1 mg/ml. Suitable aliquot of the filtered solution was added to the volumetric flask and made up to the mark with mobile phase to yield the final concentration of 25 µg/ml. Then 20 μl of this solution was injected in to column and chromatogram was recorded. The analysis was repeated in triplicate. The possibility of excipient interference in the analysis was studied.

Assay of Infusion and Eye Drop:

To determine the content of gatifloxacin in infusion and eye drop (label claim: 2 mg/ml, 3 mg/ml, respectively), aliquot of the infusion and eye drop equivalent to 10 mg was taken and transferred in to 10 ml volumetric flask separately. Then the procedure described in section 2.7.1 was followed.

Forced Degradation Studies of Bulk Drug and Pharmaceutical Formulations:

In order to determine whether the analytical method and assay were stability indicating, active pharmaceutical ingredient (API) and pharmaceutical formulations of gatifloxacin sesquihydrate were stressed under various conditions to conduct forced degradation studies.

For API and pharmaceutical formulations of gatifloxacin sesquihydrate, stock solution of gatifloxacin (1 mg/ml) was prepared separately in methanol as previously described. These stock solutions were used for forced degradation studies to provide an indication of the stability-indicating property and specificity of proposed method.

Table 5. ^aRobustness evaluation by HPLC method for GS

^b Factor	Level	Retention time (t_R) of GS (min.)	Asymmetric factor of GS peak
A: flow rate (ml/min)
0.75	-1	7.201	1.210
0.8	0	7.194	1.218
0.85	1	7.185	1.215
Mean ± S.D. (n=3)		7.193 ± 0.008	1.214 ± 0.004
B: percentage of acetonitrile in mobile phase
23	-1	7.188	1.212
25	0	7.194	1.218
27	1	7.191	1.209
Mean ± S.D. (n=3)		7.191 ± 0.003	1.213 ± 0.004
C: solvents of different Lots
First lot		7.194	1.218
Second lot		7.192	1.220
Mean ± S.D. (n=3)		7.193 ± 0.001	1.219 ± 0.001

^aAverage of three concentration (0.5, 45, 100 µg/ml), three replicates each,^b Factors were slightly changed at three levels (1, 0, -1); each time a factor was changed from level (0), the other factors remained at level

Table 6.Assay

Formulations	Labeled amount	^aAmount found ± S.D., % RSD	% Assay
Tablet (Gatiquin 400)	400 mg/tab	400.07 ± 1.33, 0.33	100.01 %
Infusion (Gatiquin)	2 mg/ml	1.99 ± 0.01, 0.56	99.63 %
Eyedrop (Gatiquin)	3 mg/ml	2.98 ± 0.01, 0.41	99.45 %

^amean concentration of three determinations.

Figure 2 Resultant HPLC chromatograms following the analysis of GS in (A) tablet (25 µg/ml), (B) infusion (25 µg/ml), (C) eye drop (25 µg/ml).

(0)

Preparation of Acid, Base and Hydrogen Peroxide Induced Degradation Product:

To 10 ml methanolic stock solution of gatifloxacin API and different pharmaceutical formulations, 10 ml of 5 M HCl, 5 M NaOH and 3 % H₂O₂ were added separately. These mixtures were refluxed separately for 4 hours at 80ºC. The forced degradation in acid, base and hydrogen peroxide media was performed in the dark in order to exclude possible degradation effect of light.

The degradation samples were then cooled to room temperature. Suitable aliquot of resultant degradation samples were taken and subjected to analysis after suitable dilutions with mobile phase.

Dry Heat- Induced Degradation Product:

For preparing dry heat degradation product, API and tablet were placed in oven at 80ºC for 24 hours under dry heat condition in the dark and then cooled to room temperature. Degradation samples were subjected to analysis after suitable dilutions with mobile phase.

Wet Heat- Induced Degradation Product:

The stock solution of gatifloxacin API and different pharmaceutical formulations were refluxed for 4 hours at 80ºC for wet heat degradation. The degradation samples were then cooled to room temperature. Suitable aliquot of resultant degradation samples were taken and subjected to analysis after suitable dilutions with mobile phase.

Photochemical Degradation Product:

The photochemical stability of drug was studied by exposing stock solution of gatifloxacin API to direct sunlight for 3 days (from 10:00 am to 06:00 pm) on wooden plank and kept in day-light. Degradation samples were subjected to analysis after suitable dilutions with mobile phase

The photochemical stability of drug in different formulations were studied by exposing formulations to direct sunlight for 3 days (from 10:00 am to 06:00 pm) on wooden plank and kept in day-light. Degradation samples were subjected to analysis after suitable dilutions with mobile phase.

Estimation of Gatifloxacin in Human Plasma:

Stock Solutions

Stock solution of gatifloxacin was prepared as described in section 2.4.

Stock solution of ciprofloxacin internal standard (I.S.) was prepared by dissolving 11.6 mg ciprofloxacin hydrochloride (equivalent to 10 mg ciprofloxacin) in 10 ml methanol. The concentration of both the stock solutions was equivalent to 1 mg/ml.

Working Stock Solutions:

Working stock solution of gatifloxacin was prepared as described in section 2.4. Four hundred μl of stock solution of ciprofloxacin was added to 10 ml volumetric flask and diluted to 10 ml with mobile phase to produce a working stock solution of 40 µg/ml.

Sample Preparation for Calibration Curve:

Aliquot ranging from 0.05-4 ml were taken from working stock solution of gatifloxacin to yield final concentration of 0.5, 1, 2, 5, 10, 20, 40 µg/ml. For this, five hundred μl of each concentration solution were placed in 2 ml micro centrifuge tubes separately. 500 μl of human plasma was added to each tube and vortexed for 30 seconds. 500 μl of I.S. working solution (containing 20 μg of I.S.) were added to each tube and vortexed for 30 seconds. 500 μl of 5 % trichloro acetic acid was added to solution and vortexed for 30 seconds. Each tube was transferred to centrifuge (Sigma Laboratory Centrifuge, Sigma, Germany) and centrifuged at 10,000 rpm for 10 min. Supernatant from all the tubes were taken and injection of 20 μl were made for each concentration. Then chromatogram was taken as described in section 2.2. The plasma calibration curve was constructed using peak area ratios of gatifloxacin to the I.S. and gatifloxacin concentrations.

Figure 3.Chromatogram of acid treated (5M HCl, 4 hours at 80˚C) GS: API: peak 1, degradant (t_R= 5.96 min.).

Figure 4. Chromatogram of base treated (5M NaOH, 4 hours at 80˚C) GS: API: peak 1, degradant (t_R= 4.85 min.).

Figure 5. Chromatogram of 3% H₂O₂ treated (4 hours at 80˚C) GS: API: peak 1, 2, 3, 4 degradants (t_R= 5.75, 13.12, 15.64, 18.08 min. respectively).

RESULTS AND DISCUSSION:

HPLC Method Development and Optimization:

Besides quantitation of GS, determination of possible degradation products is of importance during the development of pharmaceutical formulations. To analyse GS together with its possible degradation products, reverse phase LC in combination with UV detector was developed and optimization. To optimize the HPLC parameters, several mobile phase compositions were tried. It was suggested that a mobile phase containing triethylamine at acidic pH value might favor the peak shape of GS on column to achieve a reasonable retention and resolution. A satisfactory separation and peak symmetry for the drug and its degradation products were obtained with mobile phase consisting of acetonitrile and sodium acetate buffer pH 3.4 (0.2 % triethylamine was added in buffer and pH of buffer was adjusted to 3.0 with o-phosphoric acid) in the ratio of 25:75 v/v at ambient temperature with flow rate of 0.8 ml/min. Various columns were used but μBondapackTM ODS C₁₈ (300mm X 3.9mm i.d., 10μm particle size) column gave good resolution. Quantitation was achieved with UV detection at 293 nm based on peak area.

Figure 6. Chromatogram of dry heat treated (24 hours at 80˚C) GS: API: peak 1, degradant (t_R= 9.81 min.).

Figure 7. Chromatogram of wet heat treated (4 hours at 80˚C) GS: API.

Validation of Developed Stability-Indicating Method:

The results of validation of stability-indicating method developed for GS are given below.

Linearity:

The linear regression data for the calibration curve (n=3) as shown in Table 1 showed good linear relationship over concentration range 0.5-100 µg/ml. No significant difference was observed in the slopes of standard curves (ANOVA, P > 0.05).

Table 7.Forced degradation of GS in API and different pharmaceutical formulations

Sample exposure conditions	Time (hr.)	t_Rvalue of degradation products (min.)	Figure	^a% recovery (µg/100 µg) ± S.D., %RSD (n=3)
Acid, 5 M HCl, refluxed at 80ºC
API	4	5.96	Fig. 3	74.76 ± 0.38, 0.50
Tablet	4	5.95	-	74.81 ± 1.10, 1.47
Infusion	4	5.94	-	73.56 ± 0.88, 1.20
Eye drop	4	5.95	-	73.89 ± 0.64, 0.86
Base, 5 M NaOH, refluxed at 80ºC
API	4	4.85	Fig. 4	98.24 ± 0.39, 0.40
Tablet	4	5.00	-	98.00 ± 0.68, 0.69
Infusion	4	4.92	-	97.68 ± 0.43, 0.44
Eye drop	4	4.91	-	97.95 ± 0.23, 0.24
3 % H₂O₂, refluxed at 80ºC
API	4	5.75, 13.12, 15.64, 48.08	Fig. 5	44.03 ± 1.89, 4.29
Tablet	4	5.75, 13.15, 15.12	-	42.53 ± 0.73, 1.71
Infusion	4	5.75, 13.05, 15.30	-	54.91 ±1.36, 2.48
Eye drop	4	5.75, 13.03, 15.20	-	55.81 ± 2.06, 3.69
Dry heat (80ºC)
API	24	9.81	Fig. 6	96.68 ± 0.68, 0.71
Tablet	24	9.90	-	96.43 ± 1.24, 1.28
Wet heat, refluxed at 80ºC
API	4	Not detected	-	99.06 ± 0.64, 0.65
Tablet	4	Not detected	-	99.25 ± 0.80, 0.81
Infusion	4	Not detected	-	99.38 ± 0.81, 0.82
Eye drop	4	Not detected	-	98.97 ± 0.41, 0.41
Photostability
API	24	Not detected	-	99.44 ± 0.62, 0.62
Tablet	24	Not detected	-	98.94 ± 0.74, 0.75
Infusion	24	Not detected	-	99.55 ± 0.30, 0.30
Eye drop	24	Not detected	-	99.07 ± 0.45, 0.45

^a % recovery = mean measured concentration/nominal concentration (100 µg/ml) X 100.

Table 8.Precision and accuracy of GS in human plasma

Actual Concentration (µg/ml)	^aIntraday measured concentration Mean (µg/ml) ± S.D., % RSD	^bInterday measured concentration Mean (µg/ml) ± S.D., % RSD	^cAccuracy (%)
0.125	0.126 ± 0.002, 2.01	0.124 ± 0.003, 2.55	99.19
25	2.493 ± 0.014, 0.59	2.485 ± 0.006, 0.25	99.72
10	9.989 ± 0.012, 0.12	9.994 ± 0.006, 0.06	99.89

^a mean concentration of six determinations, ^bmean concentration of nine determinations, ^c% Accuracy= mean measured concentration / nominal concentration X 100.

Table 9.Stability of GS in human plasma

Nominal concentration (µg/ml)	^a Freeze-thaw stability, % drug ± S.D., % RSD	^a Solution stability, % drug ± S.D., %RSD
0.25	98.72 ± 3.25, 3.29	99.40 ± 1.17, 1.18
5	100.3 ± 0.03, 0.02	99.68 ± 0.62, 0.63

^amean percentage of three determinations.

Table 10.Summary of validation parameters

	Stability-indicating method	Human plasma method
Linearity and range
r²	0.9999	0.9996
Slope ± S.D.	75.169 ± 0.005	0.2246 ± 0.002
Intercept ± S.D.	0.114 ± 0.10	0.0024 ± 0.001
Range	0.5-100 µg/ml	0.125-10 µg/ml
LOD	0.033 µg/ml	0.041 µg/ml
LOQ	0.1 µg/ml	0.125 µg/ml
Precision (%RSD)
System precision (n=10)
Peak area ± S.D.	7517.36 ± 2.2	0.0304 ± 0.001
Retention time (t_R) ± S.D.	7.194 ± 0.0168	6.89 ± 0.004
Method precision
^aIntra-day	<1.2	<2
^bInter-day	<1.6	<2.5
^bAccuracy (mean %)	100.9 %	99.60 %

^a three concentrations, six replicates each, ^bthree concentrations, three replicates each.

LOQ and LOD:

The LOQ and LOD were determined based on a signal-to-noise ratios and were based on analytical responses of 10 and 3 times the background noise respectively. The LOQ was found to be 0.1 µg/ml with resultant % RSD of 1.2 % (n=5). The LOD was found to be 0.03 µg/ml.

Figure 8. Chromatogram of daylight treated (24 hours) GS: API.

Precision:

The % R.S.D. for repeatability of measurement of peak area was found to be 0.03 %. The measurement of concentration at three different levels showed low values of the % R.S.D. (<1.6 %) for intra- and inter-day variation, which suggested an excellent precision of the method (Table 2).

Recovery:

The proposed method when used for extraction and subsequent estimation of gatifloxacin from pharmaceutical formulations after spiking with additional drug afforded recovery of 99.99-102.40 % and mean recovery for gatifloxacin from marketed formulation are listed in Table 3.

Specificity:

The specificity of the method was established through study of resolution factors of the drug peak from the nearest resolving peak and also among all other peaks. The resolution factor for the drug peak was greater than 1.5 from the nearest resolving peak.

The specificity of LC method is illustrated in figs. 3, 4, 5 and 6 where complete separation of GS in presence of its degradation products was noticed. The average retention time ± S.D. for gatifloxacin sesquihydrate was found to be 7.194 ± 0.0168, respectively for six replicates. The peak obtained was sharp and have clear baseline separation.

Stability in Sample Solution:

Three different concentrations of gatifloxacin (0.5, 45, 100 µg/ml) were prepared from sample solution and stored at room temperature for 2 days. They were then injected in to LC system. No additional peak found in chromatogram indicating the stability of GS in the sample solution (Table 4).

Robustness:

Each factor selected (except solvents of different lots) to examine were changed at three levels (-1, 0, 1). One factor at the time was changed to estimate the effect. Thus replicate injections (n=3) of standard solution at three concentration levels were performed under small changes of three chromatographic parameters (factors). Results, presented in Table 5 indicate that the selected factors remained unaffected by small variation of these parameters. It was also found that acetonitrile of different lots from the same manufacture has no significant influence on the determination. Insignificant difference in peak areas and less variability in retention time were observed.

Assay:

The validated LC method was successfully applied for the assay of GS in different pharmaceutical formulations like tablet, eyedrop and infusion. Assay results were listed in Table 6. A typical chromatogram obtained following the assay of different pharmaceutical formulations depicted in Fig. 2.

The results of the assay indicate that the method is selective for the assay of GS without interference from excipients used in different pharmaceutical formulations.

Results of Forced Degradation Studies:

Acid, Base and Hydrogen Peroxide Induced Degradation Product:

The chromatograms of acid degraded sample for gatifloxacin showed additional peak, other than the gatifloxacin peak at retention time (t_R) value 5.9 min. (Fig.3). The chromatogram of the base degraded sample showed an additional peak at t_R value 4.9 min (Fig.4). The area of base degraded products were found to be extremely small than area of standard drug concentration (100 µg/ml) indicating that the gatifloxacin undergoes mild degradation under basic condition.

The chromatogram of the 3 % H₂O₂ degraded sample showed additional peaks at t_R values of 5.7, 13.1, 15.6, 18.08 min. (Fig.5).

Dry Heat Degradation Product:

The chromatograms of dry heat degraded sample showed additional peak at t_R value 9.9 min. (Fig.6).

Wet Heat and Photochemical Degradation Product:

The chromatograms of the sample exposed to wet heat and photochemical degradation showed no additional peaks other than the standard gatifloxacin peak (Fig. 7 and Fig. 8 respectively). This indicates that the drug is stable towards the wet heat and photochemical degradation.

The degradation of GS was found to be similar for both API and different pharmaceutical formulations.

The degradation products with their t_R values and % recovery were calculated and listed in Table 7.

Figure 9. Overlay chromatogram of gatifloxacin standards in plasma 0.125, 0.25, 0.5, 2.5, 5, 10 µg/ml.

Validation of Developed Method in Human Plasma:

Under the described conditions, gatifloxacin and the I.S. were resolved with a resolution factor greater than 3 with runtime of 10 min. The plasma calibration curve (n=3) were constructed using peak area ratio of the gatifloxacin to the I.S. and gatifloxacin concentration. The linearity over the range of 0.125-10 µg/ml was found to be quite satisfactory and reproducible over time. Three correlation co-efficient of (1) r2 = 0.9997 (2) r2 = 0.9995 (3) r2 = 0.9996 with % RSD values less than 3 were obtained following linear regression analysis. Typically, the regression equation for the calibration curve was found to be y = 0.2246 x + 0.0025, where x is the concentration in µg/ml.

Fig. 9 shows the overlay chromatogram of gatifloxacin standards in plasma 0.125, 0.25, 0.5, 2.5, 5, 10 µg/ml.

Table 8 summarizes the intra and inter-day precision and accuracy of the assay determined at gatifloxacin concentrations of the 0.125, 0.25, 10 µg/ml over three days.

The extraction recovery for gatifloxacin ranged from 75 to 87 % for the concentration 0.125 – 10 µg/ml. The extraction recovery of I.S. at a concentration 10 µg/ml was 82 %. The LOQ and LOD were determined as 0.125 µg/ml and 0.041 µg/ml respectively with 0.5 ml human plasma.

The stability of gatifloxacin in human plasma was determined using two quality control samples. The freeze and thawed stability of 0.25 and 5 µg/ml quality control samples were tested after third freeze-thawed cycle, where the first storage of 24 hours at below -20ºC was followed by two additional periods of 12-24 hours. The percent degradation was determined by comparing the mean of calculated concentration of drug from the three freeze-thawed samples with that of freshly thawed quality control samples.

The solution stability at room temperature of 0.25 and 5 µg/ml quality control samples were determined by comparing the mean of calculated concentrations from the freshly thawed quality control samples of those were kept on bench top for about 6 hours.

The results of stability of GS in human plasma are shown in table 9.

Table 10 shows the summary of validation parameters.

CONCLUSION:

A validated stability-indicating HPLC analytical method has been developed for the determination of gatifloxacin in API and different pharmaceutical formulations. The proposed method is simple, accurate, precise and specific and has the ability to separate the drug from degradation products and excipients found in the different formulations. The method is suitable for the routine analysis of gatifloxacin in API and in pharmaceutical dosage forms. (The simplicity of the method allows for application in laboratories that lack sophisticated analytical instruments such as LC/ESI-MS [8-9]). These methods are complicated, costly and time consuming rather than a simple HPLC-UV method. The developed method is also applicable for the estimation of gatifloxacin in human plasma. It may be extended to study the degradation kinetics of gatifloxacin. As method separated the drug from its degradation products, it can be employed as a stability-indicating one.

ACKNOWLEDGEMENTS:

The authors are thankful to alembic Ltd., Vadodara. for providing gift sample of gatifloxacin sesquihydrate.

REFERENCES:

1. Keam SJ, Croom KF and Keating GM. Gatifloxacin - A review of its use in the treatment of bacterial infections in the US. Drugs. 2005; 65: 695-724.

2. Blondeau JM et al. Comparative in vitro activity of gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin and trovafloxacin against 4151 Gram-negative and Gram-positive organisms. Int. J. Antimicrob. Agents. 2000; 14: 45-50.

3. Nakashima M. et al. Single- and multiple-dose pharmacokinetics of AM-1155, a new 6-fluoro-8-methoxy quinolone, in humans. Antimicrob. Agents Chemother. 1995; 39: 2635-2640.

4. Razek TMA, El-Baqary RI and Ramadan AE. Fluorimetric Determination of Gatifloxacin in Aqueous, Pure and Pharmaceutical Formulations. Analytical Letters. 2008; 41: 417-423.

5. Paramane S et al. Simultaneous RP-HPLC estimation of gatifloxacin and ornidazole in tablet dosage forms. Ind. J. Pharm. Sci. 2007; 69: 525-528.

6. Liang H, Kays MB and Sowinski KM. Separation of levofloxacin, ciprofloxacin, gatifloxacin, moxifloxacin, trovafloxacin and cinoxacin by high-performance liquid chromatography: application to levofloxacin determination in human plasma. J. Chromatogr. B. 2002; 772: 53-63.

7. Zhou HH et al. Impurity analysis and their structure determination of gatifloxacin. Yao Xue Bao. 2002; 37: 462-464.

8. Vishwanathan K, Bartlett MG and Stewart JT. Determination of gatifloxacin in human plasma by liquid chromatography/electrospray tandem mass spectrometry. Rapid Commun. Mass Spectrom. 2001; 15: 915-919.

9. Shah SA et al. A simple and sensitive HPTLC method for estimation of gatifloxacin in tablet dosage forms. Ind. J. Pharm. Sci. 2004; 66: 306-308.

10. Motwani SK et al. Stability indicating high-performance thin-layer chromatographic determination of gatifloxacin as bulk drug and from polymeric nanoparticles. Analytica Chimica Acta. 2006; 576: 253-260.

11. Ocana JA, Barragan FJ and Callejon M. Spectrofluorimetric and micelle-enhanced spectrofluorimetric determination of gatifloxacin in human urine and serum. J. Pharm. Biomed. Anal. 2005; 37: 327-332.

12. Colunga-Gonzalez LY et al. Development of a Spectrofluorimetric Method for the Determination of Gatifloxacin in Semen. Analytical Letters. 2005; 38: 2355-2364.

13. Borner K, Hartwing H and Lode H. Determination of gatifloxacin in human serum and urine by HPLC. Chromatographia. 2000; 52 (Supplement): S105-S107.

14. Dgither SA, Alvi SN and Hammami MM. Development and validation of an HPLC method for the determination of gatifloxacin stability in human plasma J. Pharm. Biomed. Anal. 2006; 41: 251-255.

15. Overholser BR, Kays MB and Sowinski KM. Determination of gatifloxacin in human serum and urine by high-performance liquid chromatography with ultraviolet detection. J. Chromatogr. B. 2003; 798: 167-173.

16. ICH Stability Testing of New Drug Substances and Products (Q1AR2), International Conference on Harmonization, IFPMA, Geneva, 2003.

17. ICH Draft Guidelines on Validation of Analytical Procedures: Definitions and Terminology. Federal Register. 1995; 60: IFPMA, Switzerland.