1. INTRODUCTION:

Prulifloxacin^1,2 6-Fluoro-1-methyl-7-[4-[(5-methyl-2–oxo-1,3-dioxol-4-yl) methyl]-1-piperazinyl]- 4-oxo-1H,4H-[1,3]thiazeto[3,2-a]quinoline-3-carboxylic acid is an orally active fluroquinolone antibacterial agent .

Prulifloxacin structure contains the skeletal quinolone with a four-member ring in the 1,2-position including a sulfur atom to increase antibacterial activity and an oxodioxolenylmethyl group in the 7-piperazine ring to improve its oral absorption; it is immediately and quantitatively transformed into the active metabolite ulifloxacin.³

PUFX showed a broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria, and several anaerobic and atypical bacteria associated to

chronic bronchitis and urinary infections⁴.

Prulifloxacin, the lipophilic prodrug of ulifloxacin, is an oral fluoroquinolone antibacterial agent with in vitro activity against various bacteria commonly associated with lower respiratory tract infections^3,5. The drug has a long elimination half-life, allowing once-daily administration.^6,7

Prulifloxacin is not official in any Pharmacopoeia and belongs to fourth-generation fluroquinolones.⁸

It is mainly used in the treatment of bronchitis exacerbation and lower urinary tract infection⁹. It shows its antibacterial activity by inhibiting DNA gyrase thus preventing DNA replication and synthesis^10,11.

Although there are a few papers published on determination of Prulifloxacin in formulation most of them deal with the assay of this constituent.^12,13 Several methods are also reported for the determination of Prulifloxacin in biological fluids.¹⁴

However the exhaustive literature survey revealed that none of the most recognized pharmacopoeias or any journals includes this drug for the determination of related substances of Prulifloxacin and the information regarding the stability of the drug is not available. So the aim of this work was to develop a liquid chromatographic procedure which will serve a reliable, accurate, sensitive and stability indicating HPLC method for the determination of related substances of Prulifloxacin in Prulifloxacin tablets.

The Regulatory agencies recommend the use of stability indicating methods (SIMs)¹⁵ for the analysis of stability samples¹⁶. This requires stress studies in order to generate the potential related impurities under stressed conditions, method development and validation¹⁷.With the evident of the International Conference on Harmonization (ICH) guidelines¹⁸, requirements for the establishment of SIMs have become more clearly mandated. The production of the potential impurities in a drug product generally take place under various environmental conditions like exposure to light, heat, hydrolysis or oxidation. Hence Stress testing can help identifying degradation products and provide important information about intrinsic stability of the drug product.

Therefore, present article reports the results of stability study of Prulifloxacin with the aim of determining the extent of the influence of different stress conditions on the stability of drug product.

2. EXPERIMENTAL:

2.1 Reagents and Materials:

Prulifloxacin active pharmaceutical ingredient (API) and test sample (Each tablet containing 600 mg Prulifloxacin) were kindly supplied by Getz Pharma Research, Ambarnath, India. Individual reference standards for Prulifloxacin impurities were not available. The EP CRS for system suitability, consisting of a mixture of all the impurities was procured from (LGC Promochem, India).

Triethylamine of Chromatographic grade was obtained from Merck Limited, Mumbai, India; Methanol was procured from Merck Mumbai, India; Acetonitrile was obtained from Rankem Mumbai, India; N,N Dimethylformamide, Ortho-Phosphoric acid, Sodium Hydroxide, Hydrochloric acid, 50% Hydrogen peroxide were obtained from Merck Limited, Mumbai, India. High purity deionised water was obtained from [Millipore, Milli-Q (Bedford, MA, USA)] purification system.

2.2 Instrumentation:

HPLC system (Waters 2695 Alliance Separation Module) equipped with inbuilt autosampler and quaternary gradient pump with an on-line degasser was used. The column compartment having temperature control and Photodiode Array/ Ultraviolet (PDA/UV) Detector (2996/2487) was employed throughout the analysis. Chromatographic data was acquired using Empower software.

The Analytical Balance used for weighing was of the make –Mettler Toledo, Model- XS205DU.

The Micro Balance used for weighing was of the make –Mettler Toledo, Model-UMX-2

The pH meter used was of the make -Thermo Electron Corp., Model-Orion-4star 1117000

2.3 Chromatographic conditions:

Kromasil C-18, 250 x 4.6 mm, 5µm (AKZO NOBEL) column was used as stationary phase maintained at 40ºC. The mobile phase involved a variable composition of solvent A (solution containing 4 mL of Triethylamine in 1000 mL water, adjusted to pH 3.4 with ortho-phosphoric acid), solvent B (Acetonitrile) and solvent C (Methanol) The mobile phase was pumped through the column with at a flow rate of 1ml min −1 (Table 1).

Table 1 Mobile phase program for gradient elution

Time in minutes	Buffer %	Acetonitrile %	Methanol %
0	70	15	15
6	70	15	15
35	35	25	40
50	35	25	40
52	70	15	15
60	70	15	15

The optimum wavelength selected was 275 nm which represents the wavelength where all impurities have suitable responses in order to permit determination of related impurities of Prulifloxacin in Prulifloxacin tablets. The stressed samples were analyzed using a PDA detector covering the range of 200–400 nm.

2.4 Solution Preparation:

2.4.1 Diluent:

The diluent used was N, N-Dimethylformamide.

2.4.2 System Suitability Solution:

The standard solution prepared was used for system suitability evaluation.

2.4.3 Standard Solution:

30.0 mg of Prulifloxacin working standard was weighed accurately and transferred into 200ml amber colored volumetric flask. 30 ml of diluent – N, N-Dimethylformamide was added and the solution was sonicated to dissolve the standard. The volume was made up to the mark with diluent. Further 2 ml of this solution was diluted to 200 ml with diluent.

2.4.4 Sample Solution:

Crushed tablet powder equivalent to 3 tablets was accurately weighed and transferred in 250 ml volumetric flask. About 200 ml of diluent N, N-Dimethylformamide was added and sonicated for 15 minutes with intermittent shaking. The solution was cooled to room temperature and the volume was made up with diluent. Further 5 ml of this solution was diluted to 50 ml with diluent and the solution was filtered through 0.45 µ Nylon filter.

2.4.5 Preparation of Placebo solution:

Prulifloxacin placebo equivalent to 3 tablets was weighed and transferred in 250 ml amber colored volumetric flask. About 200 ml of diluent N, N-Dimethylformamide was added and sonicated for 15 minutes with intermittent shaking. The solution was cooled to room temperature and the volume was made up with diluent. Further 5 ml of this solution was diluted to 50 ml with diluent and the solution was filtered through 0.45 µ Nylon filter.

2.4.6 Forced degradation sample solution for specificity study:

Multiple stressed samples were prepared as indicated below and chromatographed along with a non-stressed sample (control).

Sample stock solution:

Weighed crushed powder equivalent to 3 Tablets in 250 mL amber colored volumetric flask add about 200mL of N, N-Dimethylformamide, sonicate for 15 minutes with shaking in between, cooled and made up to the mark with the same.

2.4.6.1 Hydrolytic conditions: acid-, base-induced degradation:

Acid degradation:

5mL of sample stock solution was transferred into 50 ml amber colored volumetric flask. 2mL 0.01N Hydrochloric acid was added. Cooled at room temperature and neutralized this solution before dilution with same volume and same strength alkali. Made up the volume with diluent and the sample solution filtered through 0.45µ Nylon filter.

Base degradation:

5mL of sample stock solution was transferred into 50 ml amber colored volumetric flask. 2mL 0.01N Sodium Hydroxide was added. Cooled at room temperature and neutralized this solution before dilution with same volume and same strength acid. Made up the volume with diluent and the sample solution filtered through 0.45µ Nylon filter.

2.4.6.2 Oxidative condition: hydrogen peroxide-induced degradation:

Solution containing 0.960mgml⁻¹of Prulifloxacin was treated with 50% v/v H₂O₂under the condition shown in Table 2.

Table 2 Hydrolytic, oxidizing thermal and photolytic stress conditions

Condition	Conditions	% Degradation (% Total Impurity)	% Purity
Control	-----	1.973	98.027
Acidic	0.01N HCl	2.391	97.609
Basic	0.01N HCl	2.406	97.594
Oxidation	50% H₂O₂	13.320	86.680
Thermal	70°C for 5 Hrs	2.069	97.931
Photolytic	1.2 million lux hours/ 200Wh m^-2 UV	3.190	96.810

5mL of sample stock solution was transferred into 50 ml amber colored volumetric flask. 2mL 50% Hydrogen peroxide was added. The solution was cooled at room temperature and the volume made up with diluent. The sample solution filtered through 0.45µ Nylon filter.

2.4.6.3 Thermal degradation study:

5mL of sample stock solution was transferred into 50 ml amber colored volumetric flask. This solution was heated on the water bath at 70°C for 5 hours. Volume was made upto the mark. Filtered the sample solution through 0.45µ Nylon filter.

2.4.6.4 Photolytic degradation study:

As per guidelines for photostability testing of new drug substances and products, samples

should be exposed to light providing an overall illumination of not less than 1.2 million lx hours and an integrated near ultraviolet energy of not less than 200Wh/m2 to allow direct comparisons to be made between the drug substance and drug product¹⁹.

For photo stability testing 5mL of sample stock solution was transferred into 50 ml amber colored, 50ml clear glass and 50 ml of aluminum foil cover volumetric flask. 5 mL of N, N-Dimethylformamide was added. These solutions were exposed under UV and white light for 1.2 million lux hours. Volume was made upto the mark. The sample solution was filtered through 0.45μ Nylon filter.

The Placebo solution was treated similarly for each of the above mentioned degradation conditions.

3. RESULTS AND DISCUSSION:

3.1 Optimization of chromatographic conditions:

3.1.1 Selection of stationary phase:

Different reversed phase column were used as stationary phase selection during column selection. The column differed in length and bonding (C18) but the desired separation was achieved using Kromasil C₁₈ 250 x 4.6 mm, 5µm and thus it was proven robust in nature.

3.1.2 Influence of pH of mobile phase buffer:

A pH change of ±0.2 units did not have any adverse effect on the separation.

After optimizing various parameters, the method was finalized on Kromasil C₁₈ 250 x 4.6mm; 5µ HPLC column using variable composition of solvent A: (solution containing 4 mL of Triethylamine in 1000 mL water, adjusted to pH 3.4 with ortho-phosphoric acid), solvent B (Acetonitrile) and solvent C (Methanol). The mobile phase pumped through the column at a flow rate of 1.0 ml min⁻¹ and column compartment temperature kept at 40º C. The detector response for all the components found suitable at 275 nm; hence the typical chromatogram was recorded at this wavelength. The typical HPLC chromatograms (Figure 1) represent the satisfactory separation of all components among each other.

3.2. Method validation:

The optimized RP-HPLC method was validated according to ICH guidelines²⁰. The various validation parameters that were performed are as follows: Specificity, Accuracy, Precision (Repeatability And Intermediate Precision), Linearity, Range And Robustness. System suitability features were also assessed. Solution stability and filter compatibility were also studied.

Fig No.1a. Typical HPLC Chromatogram of mixture of all components.

Fig No.1b. Typical HPLC Chromatogram of Control Sample solution.

3.2.1. System suitability test:

The system suitability test performed according to USP 30²¹. The Standard solution was injected six times into the chromatograph and the chromatograms were recorded. The relative standard deviation of the area for peak area of Prulifloxacin for six replicate injections of standard solution should not be more than 5.0 %. The USP theoretical plates for Prulifloxacin peak should not be less than 50,000 and tailing factor should not be less than 2.0.

3.2.2. Specificity:

The peak purity indices for the analytes in stressed solutions determined with PDA detector under optimized chromatographic conditions were found to be better (purity angle < purity threshold) indicating that no additional peaks were co-eluting with the analytes and evidencing the ability of the method to assess unequivocally the analyte of interest in the presence of potential interference. Baseline resolution was achieved for all investigated compounds. The FDA guidelines indicated that well separated peaks, with resolution, Rs > 2 between the peak of interest and the closest eluting peak, are reliable for the quantification²². All the peaks meet this specification, visibly confirmed in Figs 2-4.

Fig No. 2 Typical HPLC chromatograms of stressed samples treated with

(a) Acid (b) Alkali

Fig No. 3 Typical HPLC chromatograms of stressed samples treated with Peroxide.

3.2.3. Linearity and Range:

The nominal concentration of test solutions Prulifloxacin was 1.5 µgml⁻¹. The limit of any unknown Single max impurity was kept at NMT 1.0%, NMT 2.0 % for Impurity Ulifloxacin, NMT 0.3 % for Impurity-A and NMT 3.0 % for Impurity-B and NMT 5.0% for total impurities. The relative response function was determined by preparing standard solution of each component at different concentration levels ranging from lower limit of quantification to at least 200% of impurity tolerance level and that identification of impurities below lower level of quantification were not considered to be necessary unless the potential impurities are expected to be unusually potent or toxic.

Fig No. 4 Typical HPLC chromatograms of stressed samples treated with

(a) Heat (b) Light (Photolytic Degradation)

The plots of area under the curve (AUC) of the peak responses of the analytes against their corresponding concentrations fitted straight lines responding to equations. The y-intercepts were close to zero with their confidence intervals containing the origin. The correlation co-efficient (r) for Ulifloxacin, Impurity-A and Impurity-B were found to be 1, 0.9999, and 0.9999 respectively.

3.2.3.1. Determination of limit of quantification and detection (LOQ and LOD):

The linearity performed above, was used for the determination of limit of quantification and detection. Residual standard deviation (σ) method was applied and the LOQ and LOD values were predicted using following formulas (a) and (b) and established the precision at these predicted levels.

LOQ = 10 σ (a)

LOD = 3.3 σ (b)

Where,

σ = residual standard deviation of response and

S = slope of the calibration curve.

The results are tabulated in Table 3.

Table 3 Limit of Quantification, Detection

Impurities	LOD	LOQ
Prulifloxacin and its Related Impurities
Ulifloxacin	0.002% (0.013ppm)	0.005% (0.037ppm)
Impurity A	0.002% (0.017ppm)	0.007% (0.050ppm)
Prulifloxacin	0.002% (0.017ppm)	0.007% (0.047ppm)
Impurity B	0.010% (0.072ppm)	0.033% (0.239ppm)

3.2.4 Accuracy:

Accuracy was evaluated by the simultaneous determination of analytes in solution prepared by standard addition method. The experiment was carried out by spiking placebo with Prulifloxacin, Ulifloxacin, Impurity-A and Impurity-B at 4 different levels, each level in triplicate were prepared. Single placebo preparation (unspiked), 3x LOQ, 3x 50%, 3x 100% and 3x 200% spiked placebo of the limit concentration were prepared. From the amount added and the amount found, % recovery was calculated. The results obtained are summarized in Table 4.

Table 4 Recovery data

	% Mean Recovery
	Ulifloxacin	Impurity A	Impurity B
Mean recovery at LOQ Level	100 %	100 %	98.9
Mean recovery at levels of 50%, 100%, 200% of the specification level in sample	100.1 %	97.2 %	93.3

The quantification of added analyte (%weight/weight) was carried out by using an external standard of corresponding main drug prepared at the analytical concentration. Relative response factors of all related impurities were used to calculate the weight percentage of related impurities in drug product.

(Note: In routine analysis, the RRF of the related impurities that were either not tested in the method validation with unknown identities were used as 1.)

The accuracy limits were kept at 75 to 125% at LOQ levels and 80 to 120% for other levels

The experimental results revealed that approximately 93–103 % recoveries were obtained for all the investigated related compounds. Therefore, based on the recovery data the estimation of related compounds that are prescribed in this report has been demonstrated to be accurate for intended purpose and is adequate for routine analysis.

3.2.5 Method precision and ruggedness:

ICH (International Conference on Harmonization of technical Requirements for Registration of Pharmaceuticals for Human Use) considers ruggedness as the method reproducibility and intermediate precision.

During Method precision six independent sample preparations were injected. During intermediate precision the same exercise was repeated using a fresh set of samples on a separate day, on a separate instrument, using a different HPLC column serial number by a different analyst. The results of the precision are revealed in the data given in Table 5.

Table 5 Summary Data For Precision

Analyst	% Impurity of Prulifloxacin				% Total Impurities
	Ulifloxacin	Impurity-A	Impurity-B	% Single max Impurity
	RRT-0.16	RRT-0.94	RRT-1.59	RRT-1.54
I	0.539	0.021	0.691	0.479	2.186
	0.539	0.021	0.706	0.473	2.208
	0.540	0.019	0.697	0.476	2.197
	0.537	0.020	0.689	0.474	2.175
	0.544	0.019	0.689	0.476	2.185
	0.539	0.019	0.690	0.473	2.180
II	0.559	0.020	0.696	0.492	2.231
	0.552	0.021	0.707	0.491	2.234
	0.555	0.021	0.705	0.484	2.224
	0.552	0.020	0.703	0.490	2.229
	0.550	0.021	0.703	0.491	2.231
	0.556	0.020	0.705	0.494	2.239
Mean	0.547	0.020	0.698	0.483	2.210
% RSD	1.46	4.14	1.03	1.74	1.09

Acceptance criteria:

The RSD of six determinations should not be more than 10.0%.

The RSD of 12 determinations of two analysts should not be more than 10.0%.

3.2.6. Robustness:

In order to demonstrate the robustness of the method, system suitability parameters were verified by making deliberate change in chromatographic conditions, i.e. change in flow rate by ±0.1 ml min⁻¹, change in pH of the buffer by ±0.2 units, change in wavelength by ± 5nm, change in column oven temperature by ±5°C. The standard and sample was injected and the system suitability conditions and final result was monitored. The method was demonstrated to be robust over an acceptable working range of its HPLC operational conditions. Hence it was concluded that method is Robust.

3.2.7 Solution Stability:

The sample solution was kept at sample temperature for 24 hours were injected on to the HPLC. The data obtained are summarized in Table 6.

The data shows that RSD of Standard solution up to 12 hours is less than 10%.

Table 6 Stability in analytical solution for Prulifloxacin.

Condition	Standard	% Impurity
	Area	Ulifloxacin	Imp-A	Imp-B	Single max imp
					RRT-1.54
INITIAL	76618	0.539	0.021	0.691	0.479
6Hrs	75881	0.549	0.019	0.680	0.466
12Hrs	76281	0.545	0.019	0.683	0.474
18Hrs	103912	0.548	0.021	0.702	0.470
24Hrs	123056	0.542	0.020	0.703	0.473

Thus the standard solution found to be stable for at least upto 12 hours at 15°C.

The data shows that % difference up to 24 hours is ± 0.05.

Thus the sample is found to be stable for at least upto 24 hours at 15°C.

3.2.8 Filter Compatibility:

Spiked sample solution filtered through different types of membrane syringe filters (Centrifuged, Glass, Nylon, PVDF and Teflon) were injected on HPLC. The % difference was calculated against centrifuged sample solution. The data obtained is summarized in Table 7.

Table 7 Data for Filter compatibility

Condition	% Impurity
Condition	% Ulifloxacin	% Impurity -A	% Impurity B	% Single max Imp at RRT-1.54
Centrifuged	0.541	0.019	0.719	0.478
Glass	0.542	0.022	0.695	0.475
Nylon	0.539	0.021	0.691	0.479
Nylon+Glass	0.546	0.020	0.710	0.477
Teflon+Glass	0.546	0.021	0.686	0.472
PVDF	0.548	0.021	0.701	0.473

The data shows that % Difference against centrifuged is within the limit ± 0.05.

4. CONCLUSION:

A stability study was carried out and an efficient HPLC method for the quantification of related substances of Prulifloxacin in drug product was developed and validated. The results of the stress testing of the drug, undertaken according to the ICH guidelines, revealed that the degradation products were formed in oxidative condition.

Validation experiments provided proof that the HPLC analytical method is linear in the proposed working range as well as accurate, precise (repeatability and intermediate precision levels) and specific, being able to separate the main drug from its degradation products. The proposed method was also found to be robust with respect to flow rate, column oven temperature, pH of mobile phase. Due to these characteristics, the method has stability indicating properties being fit for its intended purpose; it may find application for the routine analysis of the related substances of Prulifloxacin in Prulifloxacin tablets.

5. REFERENCES:

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Received on 24.12.2010 Modified on 09.01.2011

Research J. Pharm. and Tech. 4(9): Sept. 2011; Page 1379-1385