Stability Indicating Square-Wave Stripping Voltammetric Method for Determination of Gatifloxacin in Pharmaceutical Formulation and Human Blood

Um método de voltametria de re-dissolução catódica adsortiva de onda quadrada, indicativa de estabilidade, completamente validado, sensível e preciso foi desenvolvido para determinação de gatifloxacina na forma bruta, em formulações farmacêuticas e em amostras de plasma e soro humano. Os limites de detecção encontrados de gatifloxacina na forma bruta e no soro humano foram 1,5 × 10 e 2,2 × 10 mol L, respectivamente. O método descrito foi aplicado com sucesso na determinação de gatifloxacina em formulações farmacêuticas e em amostras biológicas humanas sem extração prévia às análises. Interferências de excipientes comuns, de alguns íons de metais comuns, de espécies orgânicas, de fármacos co-administrados e de produtos de degradação induzida por ácido não foram significativos, durante a análise de gatifloxacina nas diversas amostras analisadas. Além disso, parâmetros farmacocinéticos de gatifloxacina em plasma de voluntários saudáveis, após a administração de uma dose oral (400 mg de gatifloxacina) foram também estimados por meio do método voltamétrico de re-dissolução descrito.

So, the aim of the present work was to develop a fully validated stability-indicating square-wave adsorptive cathodic stripping voltammetic method for determination of gatifloxacin in pharmaceutical formulation and human blood without extraction prior to the analysis and without significant interferences from inorganic and organic species.

Experimental
Instruments Computer-controlled Electrochemical Analyzers Models 263A and 394-PAR (Princeton Applied Research, Oak Ridge, TN, USA) with the software package 270/250-PAR were used for the voltammetric measurements.An electrode assembly (303A-PAR) incorporated with a micro-electrochemical cell and a three-electrode system comprising of a hanging mercury drop electrode (HMDE) as a working electrode (surface area = 0.026 cm 2 ), an Ag/AgCl/KCl s reference electrode and a platinum wire auxiliary electrode, was used.A magnetic stirrer (305-PAR) and a stirring bar were used to provide the convective transport during the accumulation step.

Materials and solutions
(i) A stock standard solution of 1.0 × 10 -3 mol L -1 bulk gatifloxacin (Bristol-Myers Squibb Company, New York-Cairo) was prepared in ethanol (Merck), and then stored at 4 o C. Working solutions of gatifloxacin (1.0 × 10 -6 to 1.0 × 10 -4 mol L -1 ) were prepared daily by appropriate dilution with ethanol just before use.(ii) Ten tequin ® tablets (Bristol-Myers Squibb Company, New York-Cairo) labeled to contain 400 mg gatifloxacin per tablet were quantitatively weighed and the average mass per tablet was determined.The tablets were then grounded in a mortar to a homogeneous fine powder.A quantity of this powder equivalent to the weight of one tablet was accurately transferred into a 100.0 mL volume calibrated flask containing 70 mL ethanol (Merck).The content of the flask was sonicated for about 10 min and then filled up with ethanol.The solution was then filtered through a 0.45 µm Milli-pore filter (Gelman, Germany).The desired concentrations of the gatifloxacin were obtained by accurate dilution with ethanol.(iii) Serum sample of a healthy volunteer was stored frozen until assay.A 1.0 mL volume of the human serum was transferred to each of 10 centrifugation tubes (3.0 mL volume polypropylene micro-centrifuge tubes), containing a certain concentration of gatifloxacin (1.0 × 10 -6 to 1.0 ×10 -4 mol L -1 ), and, then, mixed well with 1.0 mL of ethanol to denature and precipitate proteins.The solutions were centrifuged (using an Eppendorf centrifuge 5417C, Hamburg, Germany) for 3 min at 14000 rpm to separate out the precipitated proteins.The clear supernatant layers of the solutions were filtered through 0.45 µm Milli-pore filters to produce protein-free human serum samples spiked with various concentrations of gatifloxacin (1.0 ×10 -6 to 1.0 ×10 -4 mol L -1 ).(iv) A series of Britton-Robinson (B-R) universal buffers of pH 2.0 to 11.5 as supporting electrolytes was prepared.A pH-meter (Crison, Barcelona, Spain) was used for the pH measurements.Deionized water was supplied from a Purite-Still Plus de-ionizer connected to an AquaMatic double-distillation water system (Hamilton Laboratory Glass LTD, Kent, UK).All the chemicals used were of analytical-grade reagents.
Induced degradation of gatifloxacin 10.0 mL of HCl solution (5.0 mol L -1 ) or 10.0 mL of NaOH solution (5.0 mol L -1 ) were mixed with 10.0 mL of ethanolic gatifloxacin solution (2.5 × 10 -3 mol L -1 ), and then subjected to forced degradation by refluxing in the dark in two-necked round-bottomed flask (50 mL volume) for 8 h.At adequate time intervals, certain volume of each of the refluxed solutions was withdrawn with micropipette to a glass tube and immediately cooled in ice path then diluted with ethanol to give a final concentration of 1.0 × 10 -4 mol L -1 gatifloxacin.A known volume (50.0 µL) of each of these solutions was introduced into a 10.0 mL volume calibrated flask then filled up with a B-R universal buffer of pH 7 to give a final concentration of 5.0 × 10 -7 mol L -1 gatifloxacin (Final pH was adjusted to pH 7 with 0.01 mol L -1 NaOH or 0.01 mol L -1 HCl).Recoveries based on three replicate measurements of gatifloxacin in each treated sample were estimated by means of the described SW-AdCS voltammetric method.

Pharmacokinetic studies
A pharmacokinetic study was performed on plasma samples of two healthy volunteers.The two volunteers gave their written informed consent prior to anticipating in the study at Ramadan Specialized Hospital (Tanta City, Egypt).They were caffeine and alcohol free and avoided all medication for at least 24 h before and during study.Each volunteer received an oral single dose of tequin® tablet (400 mg gatifloxacin).A venous blood sample (1.0 mL each) was obtained immediately before drug administration to serve as a blank (baseline) and then the sampling was continued at 0.5, 1, 1.5, 2, 3, 4, 6, 8, 12 and 24 h after the administration of the oral dose.The blood samples were centrifuged immediately at 2000 rpm for 10 min and the plasma fractions were rapidly separated and stored in coded polypropylene tubes at -20 °C until assayed.Following precipitation of proteins by ethanol and then separation by centrifugation, the plasma samples were analyzed using the described voltammetric method.

Cyclic voltammetric studies
Cyclic voltammograms of 1.0 ×10 -4 mol L -1 gatifloxacin recorded at the HMDE in the Britton-Robinson (B-R) universal buffer of pH values 3.5 to 8 exhibited a single 2-electron irreversible cathodic peak.Its peak current decreased gradually upon the increase of pH of the medium until its disappearence at pH > 10, while a second ill-defined cathodic peak appeared at more negative potentials, in solutions of pH values ≥ 8.5, Figure 1.This behavior may be attributed to the reduction of the C=O double bond of reactant species in the acidic and basic solutions.No peaks were observed in the anodic scan; pointing to the irreversible nature of the reduction process.The 2 nd cathodic peak (pH ≥ 8.5) was ill-defined due to its overlapping with that of the supporting electrolyte; therefore it can not be used for any subsequent studies.
As shown in Figure 1, the i p versus pH plot of the 1 st peak (pH < 9) is a Z-shaped curve (recalls a dissociation curve) indicating the presence of an acid-base equilibrium in the reduction of gatifloxacin. 22The pH value corresponding to the half-height of the i p versus pH plot or to the peak of δi p / δpH versus pH plot of the 1st peak (Figure 2) was found to be 6.2.This value (pH = pK a = 6.2) agrees well with those reported in the literature for pK a1 of gatifloxacin and different fluoroquinolones (6.19 and 6.03) 22,31 which is due to dissociation of their carboxylic group.On the other hand, the peak potential E p of the 1 st peak shifted to more negative values with the increase of pH, denoting that protons are involved in the electrode reaction process and the proton-transfer reaction precedes the electron transfer process. 42As shown in Figure 3, E p versus pH plot of the 1 st peak (pH < 9) exhibited two segments intersected at pH 6.2 which can be considered again to equal the first equilibrium constant of gatifloxacin. 22,31 Effect of the scan rate ν (20 to 500 mV s -1 ) on either the peak potential (E p ) and peak current (i p ) of the 1 st peak was examined at different pH values (pH < 9).The peak potentials (E p ) were linearly shifted to more negative values with the increase of scan rate (ν) confirming the irreversible nature of the reduction processes of gatifloxacin at HMDE. 43 Linear E p versus log ν plots 44 of slope values of 52 to 66 mV were obtained; from which values of αn a (product of transfer coefficient α and number of electrons n a transferred in the rate-determining step) were estimated and found to equal 1.14 to 0.89.Since, the number of electrons n a , transferred in the rate-determining step of the electroreduction of the C=O double bond of the analyte equals 2 (n a = 2) 42 the transfer coefficient α should be 0.57 to 0.45.
Linear Randles-Sevcik plots (i p versus ν) were obtained for the 1 st peak at different pH values < 9 with slope values 5.9 to 6.5 µA V -1 s (r = 0.998 ± 0.002 and n = 5) indicating that the reduction process of gatifloxacin at the HMDE is controlled by adsorption. 45,46This finding was confirmed by the linear plots of log i p versus log ν, obtained for the 1 st peaks (pH < 9) with slope values of 0.78 to 0.92 µA mV -1 s; ( r = 0.999 ± 0.001, n = 5) which are close to the theoretical values of 1.0 expected for the electrode reaction of surface species. 47he interfacial adsorptive character of gatifloxacin onto the HMDE was identified by means of cyclic voltammetry in the B-R universal buffer.Figure 3 shows the cyclic voltammograms of 5.0 × 10 -7 mol L -1 bulk gatifloxacin recorded in a B-R universal buffer of pH 7 following preconcentration onto the HMDE by adsorptive accumulation at open circuit conditions (curve a), and then at -0.7 V for 60 s (1 st cycle b and 2 nd cycle c).A better enhanced peak current magnitude was achieved following preconcentration of gatifloxacin by adsorptive accumulation onto the electrode surface (Figure 4, 1 st cycle b) whereas the 2 nd cycle exhibited a lower peak current magnitude (Figure 4, 2 nd cycle c).This behavior indicated the interfacial adsorptive character of gatifloxacin onto the mercury electrode surface.
The electrode surface coverage G 0 (amount of reactant adsorbed onto the mercury electrode surface, mol cm -2 ) was calculated using the relation: G 0 = Q / nFA, where Q is the amount of charge consumed by the surface process as calculated by the integration of the area under the peak of the cyclic voltammogram corrected for residual current, n is the total number of electrons consumed in the reduction process (n = 2) and A is the mercury electrode surface area (0.026 cm 2 ).On dividing the number of coulombs transferred 1.23 µC by the conversion factor nFA (5017.30× 10 6 µC), a surface coverage of 2.45 × 10 -10 mol cm -2 was estimated.Thus, each adsorbed gatifloxacin molecule occupies an area of 0.68 nm 2 .

Stripping voltammetric studies
Square-wave adsorptive cathodic stripping voltammograms of 5.0 ×10 -7 mol L -1 gatifloxacin recorded following its preconcentration by adsorptive accumulation onto the HMDE at -0.5 V for 30 s exhibited a single irreversible cathodic peak in the B-R universal buffer of pH values < 8.5; a better developed and sharper peak was obtained at pH 7. Therefore a B-R universal buffer of pH 7 was used as a supporting electrolyte in the rest of the present analytical study.Effect of square-wave pulse parameters (frequency f (10 to 100 Hz), scan increment ΔE s (2 to 10 mV) and pulse-amplitude a (10 to 50 mV)) on the peak current magnitude of 5.0 ×10 -7 mol L -1 gatifloxacin in a B-R universal buffer of pH 7 following its preconcentration by adsorptive accumulation onto the HMDE at E acc.= -0.5 V for 30 s was studied.A better developed peak current magnitude was achieved at f = 80 Hz, ΔE s = 10 mV and a = 25 mV.
On the other side, effect of accumulation potential (E acc. ) on the SW-AdCS voltammetric peak current magnitude of 5.0×10 -7 mol L -1 gatifloxacin in a B-R universal buffer of pH 7 was examined over the range 0.0 to -1.1 V (vs.Ag/ AgCl/KCl s ) following its preconcentration by adsorptive accumulation onto the HMDE for 30 s.A better enhanced peak current magnitude was achieved over the potential range -0.5 to -0.9 V; hence, an accumulation potential of -0.7 V was chosen throughout the present study.Effect of preconcentration time (t acc. ) at -0.7 V on the peak current magnitude for various concentrations of bulk gatifloxacin (5.0 ×10 -7 , 1.0 ×10 -7 and 1.0 ×10 -8 mol L -1 ) in a B-R universal buffer of pH 7 was also evaluated.As shown in Figure 5, for 5.0 ×10 -7 , 1.0 ×10 -7 and 1.0 ×10 -8 mol L -1 gatifloxacin the response was linear up to 40, 100 and 120 s, respectively.This means that the lower the concentration of the analyte, the longer of the accumulation duration is.Thus, the accumulation duration of choice will be dictated by the sensitivity needed.So, accumulation times of 100, 60 and 30 s were applied in the present work (Table 1).
To allow formation of a uniform distribution of the accumulated analyte onto the mercury surface, rest time was employed between the preconcentration and stripping steps.The influence of the rest time of 5.0 and 10.0 s on reproducibility of the peak current was studied and a rest time period of 5.0 s was chosen for the present analytical study, since at which sharp and better enhanced peak current was obtained.
Accordingly, the optimum operational conditions of the described stripping voltammetric method for assay of gatifloxacin were: E acc = -0.7 V, t acc ≤ 120 s, f = 80 Hz, ΔE s = 10 mV, a = 25 mV and a B-R universal buffer of pH 7 as a supporting electrolyte.

Validation of the method
Validation of the described stripping voltammetric method for assay of bulk gatifloxacin was examined via evaluation of the linear dynamic range, limit of detection (LOD), limit of quantitation (LOQ), repeatability, reproducibility, precision, robustness and intermediate precision.Linear calibration plots over various concentration ranges of bulk gatifloxacin, at various accumulation durations were obtained and the characteristics of these plots are reported in Table 1.Values of LOD and LOQ were estimated using the expression: k S.D./ b, 48 where k = 3 for LOD and 10 for LOQ, S.D. is the standard deviation of the blank (or the intercept of the calibration plot) and b is the slope of the calibration plot.The results reported in Table (1), indicated the reliability of the described SW-AdCS voltammetric method for the trace assay of bulk gatifloxacin.
The repeatability, reproducibility, precision and accuracy 49 of analysis using the described stripping voltammetric method were identified by performing four replicate measurements for each of 5.0 × 10 -8 and 1.0 × 10 -7 mol L -1 bulk gatifloxacin (t acc.= 100 s and E acc.= -0.7 V) over one day (Intra-day assay) and for three successive days (Inter-day assay).Satisfactory mean percentage recoveries (%R) for intra-and interday precisions and accuracy were achieved (Table 2).No significant differences were observed between the amounts taken and found of gatifloxacin which indicated the reproducibility, precision and accuracy of the described voltammetric method for assay of gatifloxacin.
For the study of the interference from excipients usually present in gatifloxacin formulation, 1.0 × 10 -7 mol L -1 standard bulk gatifloxacin solution and then a standard solution of tequin ® tablets containing 1.0 ×10 -7 mol L -1 gatifloxacin were analyzed following preconcentration by adsorptive accumulation onto the HMDE at -0.7 V for 100 s in both cases.No significant differences in percentage recoveries or the relative standard deviations achieved in the absence (99.88 ± 0.94) and in the presence (99.59 ± 1.06) of excipients.Thus, no significant interference from the excipients to the assay of gatifloxacin was observed by the described electroanalytical method.
Since the robustness 49 of results of an analytical method is the ability to remain unaffected with some limited changes of the operational conditions, analysis of 1.0 × 10 -7 mol L -1 bulk gatifloxacin by means of the described method was carried out under various conditions (pH 7 to 7.5, accumulation potential = -0.5 to -0.7 V, and accumulation duration = 90 to 100 s).The obtained mean percentage recoveries (99.88 ± 0.94 to 98.45 ± 0.88) were insignificantly affected within the studied conditions, and consequently the described SW-AdCS voltammetric method was reliable for assay of bulk gatifloxacin and it could be considered robust. 49he inter-laboratory precision of the measurements using the described method was examined by assay of 1.0 × 10 -7 mol L -1 gatifloxacin using two PAR-Potentiostats-Models 263A, Lab.(1) and 394, Lab.(2) under the same operational conditions at different elapsed times by two different analysts.The mean percentage recoveries obtained due to Lab. 1 (99.88 ± 0.94) to Lab. 2 (98.50 ± 0.88) and even day to day (99.10 ± 0.25 to 98.42 ± 0.65) were found reproducible, since there is no significant difference between the recoveries or relative standard deviations.

Acid-induced degradation studies
Stability testing has emerged in the field of pharmaceuticals as being very important to maintain the efficacy of a drug, as it provides measurements of the storage capacity of a drug, as well as demonstrates the safety of the drug products.Also, stability testing is a requirement for the regulatory approval during product marketing, and is a vital component of the overall quality control program.The International Conference on Harmonization (ICH) guideline entitled 50 'Stability testing of new drug substances and products' requires that stress testing be carried out to elucidate the inherent stability characteristics of the active substance.Susceptibility hydrolysis of analyte under acidic or basic conditions is one of the most required tests.An ideal stability-indicating method is one that quantifies the standard drug alone and also resolves its degradation products.Therefore, it was thought necessary to develop a simple and accurate stability-indicating method for the determination of gatifloxacin in the presence of its degradation products.
SW-AdCS voltammogram of 5.0 × 10 -7 mol L -1 of the gatifloxacin solution treated for different time intervals with 2.5 mol L -1 HCl for 8 h (as described in the experimental section), recorded at the HMDE in a B-R universal buffer of pH 7 showed a new less intense cathodic peak at less negative potential (E p = -1.20 V) in addition to the mean cathodic peak of gatifloxacin (E p = -1.45V), Figure 6.The peak current magnitude of the mean cathodic peak (2 nd peak) showed a gradual decrease with time (t) of degradation, while that of the new peak (1 st peak) increased in the same direction.This behavior indicated that gatifloxacin undergoes significant degradation under forced acidic conditions.A suggested degradation reaction is shown in Scheme 2 which is based on the hydrolysis of gatifloxacin through the cleavage of the C 7 -N 1 bond 51 and decarboxylation at C 3 -COOH bond 52 that resulted in two products of degradation (II and III).
The suggested sites of degradation were confirmed by the AM1 semi-empirical molecular orbital calculations of the bond orders throughout gatifloxacin molecule.The calculations revealed that the weakest bond orders within the gatifloxacin molecule are C 7 -N 1 (0.9941) and C 3 -COOH (0.9777).The suggested product 2-methyl- piperazine (II) is electro-inactive at the mercury electrode, while product (III), 6-fluoro-7-hydroxy-8methoxy-1-cyclopropyl-4-oxo-quinoline, is electroactive.The reduction along the 1 st peak (E p = -1.20 V) and 2 nd peak (E p = -1.45V) corresponds to reduction of the C=O double bond of the degraded product (III) and that of gatifloxacin (I), respectively.The C=O double bond of the degraded product (III) is more susceptible to reduction (E p = -1.20 V) than that of gatifloxacin (I) (E p = -1.45V) which may be due to the steric hindrance on the latter.On the other side, voltammograms of gatifloxacin solution treated with 2.5 mol L -1 NaOH showed no additional reduction peak indicating enough stability of gatifloxacin in alkaline medium.Mean percentage recoveries (%R) of gatifloxacin in its treated solutions with HCl were estimated by means of the described SW-AdCS voltammetric (Table 3).

Degradation kinetics
Concentrations of the unchanged gatifloxacin and consequently the percentage recoveries at different time intervals (t) of treatment with HCl were estimated by means of the described stripping voltammetric method using the calibration plot method (Table 3).The achieved recovery of gatifloxacin within the level of about 10% suggested a significant degradation of gatifloxacin during the forced treatment in the acid solution.Variation of concentration (C t ) of gatifloxacin with time (t) of degradation is exponential indicating first-order kinetics.A linear-fit relationship between ln (C o /C t ) and time t was obtained up to 360 min (Figure 6, Insert), which means that degradation process of gatifloxacin follows pseudo first-order kinetics.From slope value of ln (C o /C t ) versus time t plot, the apparent degradation rate constant (k) and half-life (t 1/2 ) were estimated and found to be 6.3×10 -3 min -1 and 115.5 min, respectively.

Applications Assay of gatifloxacin in presence of its degradation products
Stability indicating property of the described SW-AdCS voltammetric method was studied.Figure 7 illustrates SW-AdCS voltammograms of 5.0 × 10 -7 mol L -1 gatifloxacin solution after forced acid degradation for 480 min (curve a) and that of the same degraded solution to which 5.0 × 10 -8 mol L -1 standard gatifloxacin solution was added (curve b).As shown in Figure 7, the cathodic peak of standard gatifloxacin and that of its degradation product are well-resolved.Moreover, the percentage recovery of the added standard gatifloxacin was found to be 102.18± 0.98 indicating the successful assay of gatifloxacin without interference from its acid-induced degradation products Therefore, the method could be used successfully as a stability-indicating method for assay of gatifloxacin.

Assay of tequin ® tablets
The described SW-AdCS voltammetric method was successfully applied to assay of gatifloxacin in tequin ® tablets solution, using the calibration plot method, without the necessity for samples pretreatment and /or time-consuming extraction steps prior to analysis (Table 4).The validity of the described method was further assessed by applying the standard addition method 53 for three different standard gatifloxacin solutions added to pre-analyzed tablet solutions.The analysis exhibited also satisfactory results, which were statistically compared with those, obtained by a reported Scheme 2 Table 3. Main percentage recovery (% R) of 5.0×10 -7 mol L -1 gatifloxacin solution, at different time (t) intervals of forced degradation by refluxing with 2.5 mol L -1 HCl solution, using the described SW-AdCS voltammetric method; B-R universal buffer of pH 7; t acc = 30     *For 5% signal error.

Figure 1 .
Figure 1.Cyclic voltammograms of 1.0×10 -4 mol L -1 gatifloxacin solution in B-R universal buffer of different pH at a scan rate of 100 mVs -1 .

Figure 2 .
Figure 2. Plots of i p versus pH (curve a) and δi p / δpH versus pH (curve b) of 1.0×10 -4 mol L -1 gatifloxacin in B-R universal buffer of various pH values at a scan rate of 100 mVs -1 .

Figure 3 .
Figure 3. E p versus pH of the medium for 1.0×10 -4 mol L -1 gatifloxacin solution at a scan rate of 100 mVs -1 .

Figure 4 .
Figure 4. Cyclic voltammograms of 5.0×10 -7 mol L -1 bulk gatifloxacin in a B-R universal buffer of pH 7.0 at a scan rate of 100 mVs -1 recorded following preconcentration by adsorptive accumulation onto the HMDE at open circuit conditions (a) and then at E acc.= -0.7 V for 60 s (1 st cycle b and 2 nd cycle c).

Table 1 .Table 2 .
Characteristics of the calibration curves of SW-AdCS voltammetric determination of bulk gatifloxacin in a B-R universal buffer of pH 7; E acc.= -0.7 V, f = 80 Hz, ΔE s = 10 mV and a = 25 mV, at 25 o Analytical precision and accuracy of bulk gatifloxacin determination by the described SW-AdCS voltammetric method (n = 4) in a B-R universal buffer of pH 7; t acc = 100 s C Taken / (mol L-1

Figure 9 .
Figure 9. Mean plasma concentration-time profiles for two subjects following an oral administration of a single dose of Tequin ® tablet (400 mg gatifloxacin).

Table 6 .
s (n = 3) Estimated plasma pharmacokinetic parameters of two subjects following an oral administration of a single dose (Tequin ® tablet, 400 mg gatifloxacin)

Table 5 .
Interferences from foreign species on analysis of 2×10 -7 mol L -1 gatfloxacin by the described SW-AdCS voltammetric method