Development and Validation of A HPTLC Method for Simultaneous Densitometric Analysis of Cefixime and Potassium Clavulanate as the Bulk Drugs and in the Tablet Dosage Form

 

R.K. Nanda*, V.V. Bhagwat, S.E. Potawale and S.C. Hamane

D.Y. Patil Pratishthan’s Padmashree Dr. D.Y.Patil Institute of Pharmaceutical, Sciences and Research, Pimpri, Pune-411018, India.

*Corresponding Author E-mail: rabindrananda@rediffmail.com

 

 

ABSTRACT:

A new simple high performance thin layer chromatographic (HPTLC) method for simultaneous determination of Cefixime and Potassium Clavulanate combined tablet dosage form has been developed and validated. The separation was carried out on Merck aluminum plates precoated with silica gel 60 F254, using Acetone: Water: Acetic acid 8: 0.8: 1.2 (v/v/v) as mobile phase. The separated spots were stained with Iodine vapors and scanned at 410 nm. The retention factor for Cefixime and Potassium Clavulanate were found to be 0.23 ± 0.01 and 0.68 ± 0.01. The method was validated with respect to linearity, accuracy, precision, robustness, in accordance with ICH guidelines. The calibration curve was found to be linear over a range of 0.5–3.0 μg per spot for Cefixime and 0.310-1.870 μg per spot for Potassium Clavulanate. The method has been successfully applied for the analysis of drugs in pharmaceutical formulation. The % assay (Mean ± S.D.) was found to be 100.29± 0.9933 for Cefixime and 100.95± 1.005 for Potassium Clavulanate.

 

KEYWORDS: Cefixime, Potassium Clavulanate, HPTLC, tablet dosage form.

 

 

INTRODUCTION:

Cefixime is (6R,7R)-7-[2-(2-amino-4-thiazolyl)glyoxylamido]-8-oxo -3-vinyl-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid, [7.sup.2]-(Z)-[O-(carboxymethyl)oxime] trihydrate is officials in British Pharmacopoeia1. It is used to treat different types of bacterial infections such as bronchitis, tonsillitis, ear and skin infections, gonorrhea, and urinary tract infections2. Potassium Clavulanate is chemically (2R,3Z,5R)-3-(2-hydroxyethylidene)-7-oxa-1-azabicyclo[3.2.0]heptane-2-carboxylate. It is officials in Indian Pharmacopoeia3. Although clavulanic acid has weak antibacterial activity, it acts as a potent irreversible [beta]-lactamase inhibitor.

 

Literature survey reveals several spectroscopic4-7, HPLC 8-11 and HPTLC 12 methods for the estimation of Cefixime individually as well as in combination with other drugs. HPLC13-14 methods for the estimation of Potassium Clavulanate in combination with other drugs.

 

No reports were found for simultaneous estimation of Cefixime and Potassium Clavulanate by HPTLC method. The objective of work was to develop and validate simple, accurate, and reproducible procedure for the simultaneous HPTLC analysis of Cefixime and Potassium Clavulanate as the bulk drug and in tablet dosage forms. The proposed method is optimized and validated as per the International Conference on Harmonization (ICH) guidelines15.

 

MATERIALS AND METHODS:

Chemicals and Reagents:

Analytically pure samples of Cefixime and Potassium Clavulanate working standards were obtained as generous gifts from Hindustan Antibiotic Limited, Pimpri, Pune, India. Fixed-dose combination tablet (Hifen CV 200) containing 200 mg Cefixime and 125 mg Potassium Clavulanate were procured from Hetero HC, India. All chemicals and reagents were of analytical-grade and were purchased from Merck Chemicals, Mumbai, India.

 

Preparation of Standard stock Solutions:

Cefixime (10 mg) and Potassium Clavulanate (10 mg) were weighed separately, transferred to separate 10 ml volumetric flasks and dissolved in of methanol further diluted with methanol to furnish working standard solution of concentration 1000ng/μl for both Cefixime and Potassium Clavulanate. For analysis of the tablet dosage form, twenty tablets were weighed and their average weight was calculated.

 

Instrumentation and Chromatographic Conditions:

TLC was performed on 20 cm × 10 cm precoated silica gel 60 F254 plates (E. Merck, Germany). The plates were pre-washed with methanol, activated in an oven at 105°C for 20 min, then left to cool to room temperature. Standard solutions of Cefixime and Potassium Clavulanate were applied to the plates, as 8 mm bands 12.1 mm apart, under a stream of nitrogen, by means of a Camag Linomat V semi-automatic sample applicator fitted with a 100-μL Hamilton syringe. Plates were then developed, at constant temperature, with 20 mL Acetone: Water: Acetic acid 8: 0.8: 1.2 (v/v/v) as mobile phase, in a 20 cm ×10 cm Camag twin-trough chamber previously saturated for 20 min. The development distance was 15 cm (development time 20 min). After development the plates were removed from the chamber, dried in air. The separated spots were stained with Iodine vapors and densitometric scanning at 410 nm (Fig.1) in reflectance mode was performed with a Camag TLC Scanner-3 with winCATs software version 1.3.4 incorporating track-position optimization the slit dimensions were 6.00 mm × 0.45 mm.

 

Fig. 1: Overlain Visible spectra of Cefixime and Potassium Clavulanate.

 

Construction of calibration plots:

For preparation of calibration plots 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 μL of standard solution of Cefixime and 0.31, 0.62, 0.93, 1.25, 1.56 and 1.87 μL Potassium Clavulanate were applied to the plates which were then chromatographed and processed as described above.

 

Assay of the Marketed Formulation:

For analysis of the pharmaceutical dosage form, powder equivalent to 10 mg Cefixime (6.25 mg Potassium Clavulanate) was accurately weighed and dissolved in 10 ml methanol. The solution was centrifuged (Remi Model C30 centrifuge) for 15 min at 600 rpm then filtered through Whatman filter paper no. 41. The marketed formulation was assayed as described above. Fixed volumes of standard and sample solutions were applied to the plate which was then developed as described above. The amounts of Cefixime and Potassium Clavulanate were determined by comparing the areas obtained from the standard and sample and the amounts of Cefixime and Potassium Clavulanate per tablet were calculated.

 

RESULTS AND DISCUSSION:

A literature survey revealed that a few HPLC and HPTLC methods have been reported for estimation of Cefixime and Potassium Clavulanate combined with other drugs. The objective of the current study was to develop a precise, versatile and speedy TLC technique for determination of Cefixime and Potassium Clavulanate as the bulk drugs and in tablet dosage form.

 

Development and Validation of a Method:

Different mobile phases containing various proportions of Methanol, Water, Acetic acid, Toluene, Chloroform, Ethyl Acetate in different proportions were tried (Data not shown). The mixture of Acetone: Water: Acetic acid 8: 0.8: 1.2 (v/v/v) enabled satisfactory resolution of Cefixime and Potassium Clavulanate with good peak shape, Rf values of 0.23 ± 0.01 and 0.68 ± 0.01, respectively (Fig. 2). Pre-saturation of the TLC chamber with mobile phase for 20 min ensured good migration reproducibility of Cefixime and Potassium Clavulanate and better resolution.

 

Fig. 2: Densitogram obtained from Cefixime and Potassium Clavulanate.

 

The method was validated in accordance with ICH guidelines for linearity, accuracy, inter-day and intra-day precision, repeatability, and robustness. Linearity and Range for preparation of calibration plots for aliquots of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 μL of  working standard solution of Cefixime (1000 ng/μl) and  0.31, 0.62, 0.93, 1.25, 1.56 and 1.87 μL Potassium Clavulanate (1000 ng/μl)  were applied to the plates which were then chromatographed and processed as described under chromatographic conditions. The method was linear in the range 0.5–3.0 μg per band for Cefixime (y = 4.986x + 421.53, where y is response and x is amount; R² = 0.9955; n = 6) and 0.31-1.87 μg per band for Potassium Clavulanate (y = 4.797x + 252.85, where y is response and x is amount; R² = 0.9964; n = 6).

Table 1: Recovery studies of Cefixime and Potassium Clavulanate in the formulation Hifen CV 200.

Drug

Amount taken

(ng/band)

Amount added

(ng/band)

Total amount found

(ng/band)

% Recoveryb

(Mean ± S.D.)

Cefixime

2000

1600

3588.29

99.67 ± 1.05

2000

2000

3980.26

99.50 ± 0.19

2000

2400

4360.26

99.09 ± 0.41

Potassium Clavulanate

1250

1000

2247.03

99.86 ± 1.44

1250

1250

2478.28

99.13± 0.11

1250

1500

2714.9

98.72 ± 0.65

aFormulation Hifen CV 200, bMean± standard deviation (n=3)

 

Table 2: Results from testing of the robustness of the method for estimation of Cefixime and Potassium Clavulanate.

Condition

RSD % (n=3)

Cefixime

Potassium Clavulanate

Mobile phase composition (± 0.1 %)

1.41

1.09

Amount of mobile phase (± 0.5 %)

1.23

1.02

Time from application to  development (± 20min)

1.01

0.92

From development to scanning (± 20 min)

1.03

1.05

 

 

Accuracy:

The accuracy of the analysis was evaluated by determination of recovery at three different concentrations, equivalent to 80, 100, and 120% of the amount in the dosage form. The results indicated the method enables accurate estimation of the drugs in the tablet dosage form (Table 1).

 

Precision:

To study intra-day variation, six mixed standard solutions containing Cefixime (2000 ng/band) and Potassium Clavulanate (1250 ng/band) were prepared and applied to the plates. All solutions were analysed on the same day to record any intra-day variation in the results. To study inter-day variation, analysis of three mixed standard solutions of the same concentration was performed on three different days.

 

The intra-day and inter-day relative standard deviations were in the ranges 0.34–0.780% and 0.48–0.7924% for Cefixime and 0.75–1.549% and 0.25–0.795% for Potassium Clavulanate. These low values indicate the method is precise.

 

Repeatability of measurement of peak area was determined by applying Cefixime and Potassium Clavulanate solutions (2μL) to a TLC plate, developing the plate, and scanning the separated spots of Cefixime and Potassium Clavulanate six times without changing the position of the plate. The RSD of peak-area measurement was calculated and found to be 0.9070% and 0.8092% for Cefixime and Potassium Clavulanate respectively.

 

Robustness Studies:

The effect of small, deliberate variation of the analytical conditions on the peak areas of the drugs was examined. Factors varied were mobile phase composition (± 0.1 %) volume of mobile phase (± 0.5 %), time from application to development (± 20min) and from development to scanning (± 20 min). One factor at a time was changed to study the effect. The robustness of the method was checked at amount of 2000 and 1250 ng/band for Cefixime and Potassium Clavulanate, respectively. The RSD (%) of peak area was calculated for each change of conditions and found to be within the range stipulated by ICH guidelines     (Table 2).

 

CONCLUSION:

It is shown above that the accuracy, precision, reproducibility, repeatability, and linearity of this new TLC–densitometric method compare favourably with those of HPLC, HPTLC, spectrophotometry, and other methods reported regularly in the literature. The results also meet ICH guidelines for validation of pharmaceutical TLC methods. The proposed method is less expensive, simpler, more rapid, and more flexible.

 

ACKNOWLEDGEMENTS:

The authors express their gratitude to Dr. A. D. Deshpande, Director, Padm. Dr. D. Y. Patil Institute of Pharmaceutical Sciences and Research, Pune, MH, India, for providing necessary facilities, and to Hindusthan Antibiotic Limited, Pimpri, Pune India for the generous gift samples of pure Cefixime and Potassium Clavulanate.

 

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Received on 25.05.2010        Modified on 10.06.2010

Accepted on 24.06.2010        © AJRC All right reserved

Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 998-1001