INTRODUCTION:

Cefditoren Pivoxil is a third-generation cephalosporin antibiotic. It is having same mechanism of action as other cephalosporins¹. Cephalosporins disrupt the synthesis of the peptidoglycan layer of bacterial cell walls. The peptidoglycan layer is important for cell wall structural integrity. The final transpeptidation step in the synthesis of the peptidoglycan is facilitated by transpeptidasesknown as penicillin-binding proteins (PBPs). PBPs bind to the D-Ala-D-Ala at the end of muropeptides (peptidoglycan precursors) to crosslink the peptidoglycan. Beta-lactam antibiotics mimic this site and competitively inhibit PBP crosslinking of peptidoglycan. Chemically it is (2,2-dimethylpropanoyloxymethyl (6R,7R)-7-([(2E)-2-(2-amino-1,3-thiazol-4-yl)-2-methoxyiminoacetyl]amino)-3 -[(Z)-2-(4-methyl-1,3-thiazol-5-yl)ethenyl]-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylate.^4-5

Stability testing forms an important part of the process of drug product development. The purpose of stability testing is to provide evidence on how the quality of a drug substance or drug product varies with time under the influence of a variety of environmental factors such as temperature, humidity, and light, and enables recommendation of storage conditions, retest periods, and shelf lives to be established. The two main aspects of drug product that play an important role in shelf life determination are assay of active drug, and degradants generated, during the stability study. The assay of drug product in stability test sample needs to be determined using stability indicating method, as recommended by the International Conference on Harmonization (ICH) guidelines² and USP 26³. It is official in United state Pharmacopeia⁴ and Japanese Pharmacopeia⁵. Detailed survey of literature for CFDT revealed several methods such as Spectorphotometric methods^6,7 and Fast LC⁸ method for its determination from pharmaceuticals. This manuscript describes the development and subsequent validation of a stability indicating isocratic reversed phase HPLC method for simultaneous determination of CFDT in presence of their degradants. To establish the stability indicating nature of the method, forced degradation of drug substances and drug productwas performed under stress conditions (thermal, photolytic, acid and basic hydrolytic and oxidative), and stressed samples were analysed by the proposed method. The proposed LC method was able to separate both drugs from degradants generated during forced degradation studies. The linearity of response, accuracy, intermediate precision of the described method for assay of CFDT has been checked.

EXPERIMENTAL:

Chemicals and Reagents:

CFDT standard was obtained as a gift from Torrent Pharmaceutical (Ahmedabad, India). Drug product ( Lable claim: Cefditoren Pivoxil 200 mg ) as CEFTORIN-tab was purchased from the market. Acetonitrile, methanol, sodium hydroxide, hydrochloric acid and hydrogen peroxide were from Qualigens Fine Chemicals (Glaxo Ltd.).

HPLC instrumentation and Conditions:

A Shimadzu HPLC instrument (LC_2010 CHT) [software LC Solution, equipped with prominence diode array detector (SPD-M20A), Auto-sampler]. The chromatographic separation was performed using a Phenomenex Luna C18 (150 mm ´4.6 mm, 5 µm particle size), mobile phase consisting Acetonitrile: Water (50: 50, v/v) with apparent pH adjusted to 3.5 adjusted with Ortho-phosphoric acid, filtered through 0.45µm nylon filter and degassed in ultrasonic bath prior to use and UV detection at 230 nm using photo diode array detector. For analysis of forced degradation samples, the photodiode array detector was used in scan mode with a scan range of 220–400 nm and desired peak coverage of 100%. Peak homogeneity was expressed in terms of peak purity values, and was obtained directly from the spectral analysis report obtained using the above-mentioned software.

Standard and sample preparation:

The standard stock solutions 1000 µg/ml of CFDT was prepared by dissolving working standard in small proportion of methanol and later diluted to desired volume with mobile phase. The standard calibration solution of CFDT having concentration in the range of 20-120 µg/ml were prepared by diluting stock solution with mobile phase.

Ten tablets were weighed, their mean weight determined, and crushed in mortar. An amount of powdered mass equivalent to one tablet content was transferred into a 50ml volumetric flask containing 10 ml of methanol, mechanically shaken for 10 min, ultrasonicated for 5 min, and then diluted to volume with mobile phase (sample stock solution). 5ml aliquot diluted to 50 ml with mobile phase (sample solution). A small portion of sample solution was filtered through 0.45µm nylon filter and used for injection on HPLC.

Procedure for forced degradation study:

Forced degradation of each drug substances and the drug product was carried out under acid/base hydrolytic, Neutral and oxidative stress conditions.

For Acid hydrolysis, accurately weighed quantity of 10 mg CFDT was transferred into 10 ml volumetric flask, dissolve with 3 ml of methanol and dilute up to mark with 0.1 N HCl (1000 µg/ml). It was kept for 3 hours at room temperature. From this solution 1 ml was taken and transferred into 10 ml volumetric flask and diluted up to mark with mobile phase (100µg/ml).

For base hydrolysis, accurately weighed quantity of 10 mg CFDT was transferred into 10 ml volumetric flask, dissolve with 2 ml of methanol and dilute up to mark with 0.001 N NaOH (1000 µg/ml). It was kept for 5 minutes at room temperature. From this solution 1 ml was taken and transferred into 10 ml volumetric flask and diluted up to mark with mobile phase (100µg/ml).

For Neutral hydrolysis, accurately weighed quantity of 10 mg CFDT was transferred into 10 ml volumetric flask, dissolve with 5 ml of methanol and dilute up to mark with water (1000 µg/ml). It was kept for 12 hours at room temperature. From this solution 1 ml was taken and transferred into 10 ml volumetric flask and diluted up to mark with mobile phase (100µg/ml).

For oxidative hydrolysis, accurately weighed quantity of 10 mg CFDT was transferred into 10 ml volumetric flask, dissolve with 7 ml of methanol and dilute up to mark with 3 % H₂O₂ for 5 hours at room temperature (1000 µg/ml). After 5 hours, from this solution 1 ml was taken and transferred into 10 ml volumetric flask and diluted up to mark with mobile phase (100µg/ml).

RESULT AND DISCUSSION:

This method describes a reversed phase HPLC procedure employing a Shimadzu HPLC instrument (LC_2010 CHT) [software LC Solution, equipped with prominence diode array detector (SPD-M20A), Auto-sampler]. The chromatographic separation was performed using a Phenomenex Luna C18 (150 mm ´4.6 mm, 5 µm particle size), mobile phase consisting Acetonitrile: Water (50: 50, v/v) with apparent pH adjusted to 3.5 adjusted with Ortho-phosphoric acid, filtered through 0.45µm nylon filter and degassed in ultrasonic bath prior to use and UV detection at 230 nm using photo diode array detector.

There are total five impurities generated from each hydrolysis. (Fig. 1-4) and percentage degradation is shown in table 1. CFDT was moderately degradable under oxidative condition and highly degradable under acidic condition. In, contrast, CFDT was relatively stable under basic and neutral hydrolysis.

Table 1: Results of analysis of forced degradation study samples using proposed method, indicating percentage degradation of CFDT and purity of CFDT peak in chromatograms.

Stress condition	% Degra dation	Peak purity

Acidic hydrolysis, 0.1 N HCl, 3 hours	2.75	999
Alkali hydrolysis, 0.001 N NaOH, 5 minutes	24.02	999
Neutral hydrolysis, H₂O, 12 hours	3.68	999
Oxidation, 3 % H₂O_2, 5 hours	40.12	999

*Peak purity values between 990-1000 indicates homogeneous peak

Figure 1: Chromatogram of CFDT under Acid Hydrolysis

Figure 2: Chromatogram of CFDT under Alkali Hydrolysis

Figure 3: Chromatogram of CFDT under Oxidative degradation

Figure 4: Chromatogram of CFDT under Neutral Hydrolysis

METHOD VALIDATION:

The present method was validated for specificity, Accuracy and intermediate precision. The nominal concentrations of standard and test solutions for CFDT were 20 µg/ml. The standard solutions for linearity were prepared five times and inter-run precision for slope of regressed line was found to be 0.99% R.S.D. for CFDT. The correlation coefficients were found to be more than 0.998 for CFDT (Table 2). Accuracy and precision of the method was determined by performing the recovery experiment. Five replicates sample of each concentration level were prepared and mean assay was determined for intermediate precision which indicates method is precise (Table 4).This experiment was performed at three levels, in which sample stock solutions were spiked with standard drug solution containing 50%, 100% and 150% of labeled amount of both the drug in tablet. Three replicate samples of each concentration level were prepared and the %recovery at each level (n = 3), and mean %recovery (n=9) were determined (Table 3). The mean recovery was found to be 98.77-101.29%. Based on the peak purity results, obtained from the analysis of forced degraded samples using the described method, it can be concluded that the method is specific for estimation of CFDT in presence of degradants. The method has linear response in stated range and is accurate and precise. The described method can be used as stability indicating method for assay of CFDT in pharmaceutical dosage form.

Table 2: Method validation data for CFDT

Regression Analysis	Statistics Value
Linearity range (µg/ml)	20-120
Regression Equation	y = 40,616.84x - 24,357.77
Correlation Co-efficient	R² = 0.9994
Slope	40,616.84
Intercept	24,357.77

Table 3: Results of accuracy

Amount of CFDT in sample (µg)	Amount of Std. CFDT added	Total amount	Total amount found	% Recovery
40	0	40	40.02	-
40	20	60	59.14	98.33
40	40	80	79.12	99.23
40	60	100	100.4	101.54

Table 4: Results of Intermediate precision for CFDT assay in tablet formulation using proposed method

Concentration(µg/ml)	Mean assay±%RSD
Set 1 (n=5)	99.72±0.83
Set 2 (n=5)	98.92±0.52

ACKNOWLEDGEMENT:

The author is very thankful to Torrent Pharmaceutical Pvt. Ltd, Ahmadabad for providing the gift sample of Cefditoren Pivoxil.

REFERENCES:

1. Overington JP, Al-Lazikani B, Hopkins AL: How many drug targets are there? Nat Rev Drug Discov.5(12), 2006,993-6.

2. Frishman WH, Ram CV, McMahon FG, Chrysant SG, Graff A., Kupiec JW, Journal of Clinical Pharmacology, 35 ,1995, 1060–1066.

3. Fogari R., Corea L., Cardoni O, Cosmi F., Porcellati C, Innocenti P., Provvidenza M., Journal of Cardiovascular Pharmacology, 30, 1997, 497–503.

4. United state pharmacopoeia’s Pending monograph, Available from: October 2010 http://www.usp.org.

5. Japanese Pharmacopeia, Official monograph, The ministry of Health, Labour and Welfare , Prefectural Office in Japan ,437-38.

6. Niraimathi V., Aruna A., Suresh J., Prema V. Spectrophotometric Estimation of cefditoren pivoxil in pharmaceutical oral solid dosage form. International Journal of Chemical Science, 8, 2010, 724-8.

7. Niraimathi V, Aruna A, Suresh J, Prema V. Estimation of cefditoren pivoxil in pharmaceutical oral solid dosage form by spectrophotometry. Journal of Phharmaceutical Research, 2, 2010, 63-65.

8. Kumar N, Nageswara Rao G., Naidu P.Y. Fast LC Method for Determination of Cefditoren Pivoxil and Its Related Impurities in Bulk and Pharmaceutical Formulations. Asian journal of Research in Chemistry, 3, 2010, 943.

Received on 29.06.2011 Modified on 17.07.2011

Research J. Pharm. and Tech. 4(9): Sept. 2011; Page 1461-1464