1. INTRODUCTION:

Nadifloxacin (NAD), chemically know as (9-Fluoro-6,7-dihydro-8-(4-hydroxy piperidino)-5-methyl-1-oxo-1H, 5H-benzo [ij] quinolizine-2-carboxylic acid), is a Fluoroquinolone antibiotic used for the treatment of skin infections with susceptible bacteria, Acne vulgaris ( Inflamed lesions)(1 – 2).

Literature survey reveals HPLC determination of Nadifloxacin in rat plasma (3) & UV–visible spectroscopic studies about the interactions between polyamidoamine (PAMAM) dendrimers and quinolones (nadifloxacin and prulifloxacin)(4).No HPTLC method is reported so far for the estimation of drug in pharmaceutical formulations; hence we have developed a stability indicating HPTLC method for the estimation of nadifloxacin in bulk and pharmaceutical formulation. Structure of Nadifloxacin is shown in Figure 1.

2. EXPERIMENTAL:

2.1. Reagents and Chemicals:

Nadifloxacin was supplied as a gift sample by Cipla Ltd (Goa, India). All chemicals and reagents used were of HPLC/AR grade.

2.2. Instrumentation and Chromatographic Conditions:

The standard solutions ranging from 50-300 ng/band were applied on precoated silica gel 60 GF₂₅₄plate in the form of bands with 100 µl sample syringe using automatic sample applicator LINOMAT V. The linear ascending development was carried out in 10 cm ×10 cm twin trough glass chamber which was already saturated for 30 min. with the mobile phase. The mobile phase consists of Chloroform: Methanol: Ammonia (4.3: 4.3: 1.4v/v/v). After development plate was immediately dried with the help of dryer and was observed under UV chamber. The well resolved band of drug was scanned at 296 nm with Camag TLC scanner III densitometer controlled by WINCAT’s software. The source of radiation utilized was deuterium lamp emitting a continuous UV spectrum between 200 to 400 nm.

2.3. Standard Solutions and Calibration Graphs:

Stock solution was prepared by dissolving 100 mg of nadifloxacin in 100 mL methanol, from which 1 mL was further diluted to 100 mL with methanol to get stock solution of 10 ng/µL. The standard solutions were applied in a concentration range of 50–300 ng/band for nadifloxacin. The plate was developed on previously described mobile phase. The peak areas were plotted against the corresponding concentrations to obtain the calibration graphs.

2.4. Analysis of Formulation:

A quantity of cream equivalent to 10 mg of NAD (1 g of cream) was weighed and transferred to a 100 ml volumetric flask about 30 ml of methanol was added, just warmed 5-10 mins, ultrasonicated for 20 mins and volume was made up to the mark with the methanol. The solution was filtered using Whatman paper No. 41. From the filtrate 1 ml was further diluted to 10 ml to get sample stock solution of NAD 10 ng/µl. From sample stock solution 10 µl volumes was applied on HPTLC plate to obtain final concentration of 100 ng/band for NAD. Six sample solutions were applied and after chromatographic development peak areas of the spots were measured at 296 nm and concentrations in the samples were determined using multilevel calibration.

Figure 1. Structure of NAD

Figure 2. Standard densitogram of NAD

Figure 3. In situ spectrum of NAD standard and sample measured from 200 to 400nm

Figure 4. H₂O₂ degradation

Table 1: Linear regression data for calibration curves for NAD (n = 3)

Parameter	NAD
Detection Wavelength (nm)	296
Beer’s Law Limit (ng/band)	50-300
Regression equation	y = 26.212x + 1407.3
Correlation Coefficient (r)	0.9979
Intercept (c) ± SD	26.212 ± 0.89
Slope (m) ± SD	1407.273 ± 114.34
Limit of detection (ng/band)	14.39
Limit of quantitation (ng/band)	43.62

3. METHOD VALIDATION:

The proposed method was optimized and validated as per the International Conference on Harmonization (ICH) guidelines (5). The following parameters were validated

Table 2: Results of marketed formulation analysis

Marketed formulation

Drug

Label claim

(% w/w)

Area* of densitogram

% Mean amount estimated*± S.D*

Nadibact

NAD

4072.07

101.24 ± 1.41

*Average of six determinations

Table 3: Statistical evaluation of precision of developed method

Drug - NAD	Precision
Drug - NAD	Intraday*	Interday**
Conc.(ng/band)	100	80, 100, 120
Mean area ± SD	4069.95 ± 25.79	4051.02 ± 11.24
% Content ± SD	101.35 ± 0.97	100.56 ± 1.11
RSD (%)	0.96	1.11
S.E.	0.3967	0.3713

*Average of six determinations **Average of nine determinations

3.1. Precision:

The precision was determined at two levels, i.e. repeatability and intermediate precision. Repeatability was determined by six replicate applications of a sample solution at the analytical concentration. Inter-day variation was carried out with six determinations at 100% of test concentration and intraday variation for determination of NAD was carried out with six replicates at concentration levels 100 ng/band on different days.

Table 5: Robustness of method

A: Chromatographic Changes (% of chloroform in mobile phase)

% change in mobile phase (Methanol: Chloroform: Ammonia)	Rf	Peak area
43 : 43 : 14	0.62	4092.8
42.14 : 43.86 : 14	0.61	4046.6
43.86 : 42.14 :14	0.63	4062.7
43 : 43.28 :13.72	0.61	4050.83
43.28 : 43 :13.72	0.63	4081.7
*Mean± S.D.**	0.62 ± 0.01	4066.92 ± 19.87

*Average of three determinations

3.2. Robustness:

The robustness of the method was studied, during method development, by small but deliberate variations in mobile phase composition (± 2%), chamber saturation period (± 10%), development distance (± 10%), time from application to development (0, 10, 15, 20 min), time from development to scanning (0, 30, 60, 90 min). One factor at a time was changed at a concentration level of 100ng/band of NAD, to study the effect on the peak area of the drugs.

3.3. Limit of Detection and Limit of Quantitation:

The LOD and LOQ were separately determined based on the calibration curves.

3.4. Specificity:

The specificity of the method was ascertained by analyzing standard drug and sample. The spot for nadifloxacin in sample was confirmed by comparing the Rf and spectra of the spot with that of standard. The peak purity of nadifloxacin was assessed by comparing the spectra at three different levels, i.e., peak start (S), peak apex (M) and peak end (E) positions of the spot.

3.5. Recovery Studies:

To check the degree of accuracy of the method, recovery studies were performed in triplicate by standard addition method at 80%, 100% and 120%. A known amount of standard NAD was added to pre-analyzed samples and was subjected to the proposed HPTLC method. At each level of recovery three determinations were performed.

4. Forced Degradation Studies:

The International Conference on Harmonization (ICH) guideline entitled ‘‘Stability Testing of New Drug Substances and Products’’ requires the performance of stress testing to elucidate the inherent stability characteristics of the active substance [6]. An ideal stability indicating method is one that quantifies not only the drug compound alone but also resolves its degradation products.

B: Chromatographic Changes (chamber saturation)

Chamber saturation	Rf	Peak area
13.5 mins	0.63	4021.0
15 mins	0.62	4075.0
16.5 mins	0.62	4066.0
*Mean± S.D.**	0.62 ± 0.01	4054.0 ± 28.93

*Average of three determinations

Preparation of stock solution:

A stock solution containing 100 mg of nadifloxacin in 100 mL methanol was prepared, further diluted 1 mL of above solution up to 100 mL with methanol. This stock solution (10 μg/mL) was used for forced degradation to provide an indication of the stability indicating property and specificity of the proposed method.

4.1. Acid Degradation Studies:

To 10 mL of methanolic stock solution of nadifloxacin added 10 mL of 5 N HCl. This mixture was refluxed 24 hr. 20 µL (100 ng/spot) of the resultant solutions were applied on TLC plate and was developed and scanned under above established chromatographic conditions.

4.2. Alkali Degradation Studies:

To 10 mL of methanolic stock solution of nadifloxacin added 10 mL of 5 N NaOH. This mixture was refluxed 24 hr. 20 µL (100 ng/spot) of the resultant solutions were applied on TLC plate.

4.3. Neutral Degradation Studies:

To 10 mL of methanolic stock solution of nadifloxacin added 10 mL of water. This mixture was refluxed 24 hr. 20 µL (100 ng/spot) of the resultant solutions were applied on TLC plate and was developed and scanned under above established chromatographic conditions.

4.4. Oxidation Studies:

To 10 mL of methanolic stock solution of nadifloxacin, 10 mL of hydrogen peroxide (H₂O₂) (30%, v/v) was added. This solution was heated in boiling water bath for 10 min to remove completely the excess of hydrogen peroxide and then refluxed for 8 hr at 80 °C. 20 µL (100 ng/spot) of the resultant solutions were applied on TLC plate and was developed and scanned under above established chromatographic conditions.

4.5. Dry heat-induced Degradation:

The bulk nadifloxacin was stored at 100 °C for 24 hr under dry heat conditions. The methanolic stock solution of this dry heat exposed drug was prepared as described above and 10 mL of the prepared stock solution was diluted to 20 mL with methanol (5 μg/mL). 20 µL (100 ng/spot) of the resultant solutions were applied on TLC plate and was developed and scanned under above established chromatographic conditions.

4.6. Wet heat-induced Degradation:

10 mL of the methanolic stock solution of nadifloxacin was diluted to 20 mL with methanol (5 μg/mL) and refluxed for 3 hr at boiling water bath to study the wet heat degradation. 20 µL (100 ng/spot) of the resultant solutions were applied on TLC plate and was developed and scanned under above established chromatographic conditions.

C: Chromatographic Changes (development distance)

Development distance	Rf	Peak area
88 mm	0.62	4069.2
80 mm	0.63	4034.7
72 mm	0.64	4035.6
*Mean± S.D.**	0.63 ± 0.01	4046.5 ± 19.66

*Average of three determinations

D: Chromatographic Changes (Time from application to development)

Time from application to development	Rf	Peak area
0	0.62	4050.3
10 mins	0.64	4069.7
15 mins	0.64	4056.7
20 mins	0.65	4039.8
*Mean± S.D.**	0.64 ± 0.01	4054.1 ± 12.50

*Average of three determinations

4.7. Photochemical and UV Degradation Product:

10 mL of the methanolic stock solution of nadifloxacin was diluted to 20 mL with methanol (5 μg/mL) and the photochemical stability of the drug was studied by exposing the stock solution to direct sunlight for 4 days on a wooden plank and kept on terrace. The drug solution was also exposed to UV irradiation at 254 nm for 8 h in a UV-chamber. 20 µL (100 ng/spot) of the resultant solutions were applied on TLC plate and was developed and scanned under above established chromatographic conditions.

4.8. Detection of Related Impurities:

The related unknown impurities were determined by spotting higher concentrations of the nadifloxacin. Nadifloxacin solution was prepared of a concentration 100 μg/mL in methanol, and this solution was termed as sample solution. 1 mL of the sample solution was diluted to 10 mL with methanol and this solution was termed as standard solution (10 μg/mL). 10 µL of the sample solution (1000ng/spot) and standard solution (100 ng/spot) were applied on HPTLC plate and was developed and scanned under above established chromatographic conditions.

5. RESULTS AND DISCUSSION:

5.1. Optimization of Procedures:

While selection of mobile phase different proportions of methanol, chloroform, ammonia were tried. Ultimately mobile phase containing Chloroform: Methanol: Ammonia (4.3: 4.3: 1.4 v/v/v) was finalized as optimal for obtaining well defined and resolved peaks. The spots developed were dense, compact and typical peak of NAD was obtained. Peak was symmetrical in nature and no tailing was observed when plates were scanned at 296 nm (Figure 2).

E: Chromatographic Changes (Time from development to scanning)

Time from development to scanning	Rf	Peak area
0	0.62	4061.27
30min	0.62	4064.27
1 hr	0.62	4064.27
1 hr 30 mins	0.62	4070.17
*Mean± S.D.**	0.62 ± 0.00	4065.00 ± 3.73

*Average of three determinations

Table 6: Results of forced degradation studies

Stress condition	Time	% Assay of active substance	R_f values of degradation products
Acid hydrolysis (5N HCl)	24 hrs	100.70	No degradation
Base hydrolysis (5N NaOH)	24 hrs	100.74	No degradation
Neutral (H2O)	24 hrs	101.75	No degradation
Oxidation (30% H2O2,80°C)	8 hrs	86.72	0.59
Dry Heat (100°C)	24 hrs	97.24	No degradation
Wet Heat (On boiling water bath)	3 hrs	100.85	No degradation
Photo stability	4 days	63.46	0.49
UV radiation	8 hrs	101.05	No degradation

5.2. Method Validation

NAD showed good correlation coefficient in concentration range of 50–300 ng/band (r² = 0.9979) (Table 1). The spot at Rf 0.62 was observed in the densitogram of the drug sample extracted from cream. There was no interference from the excipients commonly present in the creams. The % assay for nadifloxacin was found to be 101.24 ± 1.40 % RSD (Table 2). The low %RSD value indicated the suitability of this method for routine analysis. Good correlation was obtained between standard and sample spectra of nadifloxacin. The comparative spectrum of standard and sample was given in Figure 3. %RSD was found to be less than 2 % for day to day variations, which proves that method was precise. Results were shown in Table 3. Results of recovery studies were shown in Table 4. Method was found robust and results were given in Table 5.

5.3. Stability-Indicating Property

HPTLC studies of the samples obtained during the stress testing of NAD under different conditions using Chloroform: Methanol: Ammonia (4.3: 4.3: 1.4v/v/v) as the mobile phase suggested the following degradation behavior (Table 6).

NAD was found to be stable in acidic, alkaline and neutral condition. Also the drug was stable in UV light for the period of 8 hr. The drug was degraded in 30% hydrogen peroxide at 80°C and shows degradation peak (D1) at Rf 0.59 as shown in Figure 4. The drug when subjected to dry heat, it does not show any degradation peak but the assay of active drug gets reduced to 97.24%. When drug was subjected to wet heat it shows broadening of peak at bottom but the area was not get hampered. In sunlight drug was get degraded and degradation peak (D2) was observed at 0.49 Rf as shown in Figure 5.

Figure 5. Photo stability

5.4. Related Impurities:

Only principal spots for nadifloxacin were observed in sample and standard solution, which indicates that drug does not show any related impurities to nadifloxacin.

6. CONCLUSION:

The proposed HPTLC method was validated as per ICH guidelines. The %RSD and standard error calculated for the method were low, indicating high degree of precision of the methods. The results of the recovery studies performed show the high degree of accuracy of the proposed methods. The results of the stress studies indicated the specificity of the method. Hence, it can be concluded that the developed HPTLC method was accurate, precise and selective and can be employed successfully for the estimation of Nadifloxacin in topical cream.

7. ACKNOWLEDGEMENT:

The authors are thankful to Dr. A.D. Despande, Director of Pharmacy, Padmashri Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune for providing necessary facilities and to Cipla Ltd (Goa, India) for providing gift sample of pure drug.

8. REFERENCES:

1. O’Neil MJ. Heckelman PE. Koch CB. Roman KJ. Kennu CM. and D’Arecca MR. Nadifloxacin. In Merck Index. John Merck research laboratories division of Merck & Co., INC. Whitehouse station, NJ, USA. 2006; 14th ed: pp. 1097-1098.

2. Jacobs MR. and Appelbaum PC. Nadifloxacin: a quinolone for topical treatment of skin infections and potential for systemic use of its active isomer, WCK 771. Expert Opin. Pharmacotherapy. 2006; 7: 1957.

3. Lunn G. and Wiley J. HPLC Methods for Recently Approved Pharmaceuticals. J. Wiley & Sons INC publication, New York, NY, USA. 2005; pp. 424.

4. Cheng Y. Qu H. Ma M. Xu Z. Xu P. Fang Y. and Xu T. European Journal of Medicinal Chemistry 2007; 42: 1032-1038.

5. ICH Q2 (R1), Harmonised Tripartite Guideline, Validation of Analytical Procedures: Text and Methodology. 2005.

6. ICH–QA1 (R2) Stability testing of new drug substance and products. International Conference on Harmonization, Geneva, February 2003.

Received on 24.12.2009 Accepted on 20.02.2010

Res. J. Topical and Cosmetic Sci. 1(1): Jan. – June 2010 page 25-29

Level of recovery (%)	Total amt of drug recovered (ng/band)	Amount of drug estimated in mg/g of cream	Amount of pure drug recovered	% Recovery*	SD	S.E.	% RSD
80	181.26	101.47	79.79	99.65	0.55	0.3164	0.55
100	200.91	101.39	99.52	99.49	0.46	0.2677	0.46
120	221.14	101.50	119.64	99.65	0.40	0.2338	0.41