INTRODUCTION:
Cefixime trihydrate, 5-thia-1-azabicyclo [4.2.0] oct-2-ene-2-corboxylic acid, 7-[[2-amino-4-thiazolyl)[(carboxymethoxy)imino]acetyl]amino]-3-ethenyl-8-oxo-,trihydrate is a broad spectrum, third generation cephalosporin antibiotic for oral administration. Cefixime kills bacteria by interfering in the synthesis of the bacterial cell wall1. It is official in USP (2007)2.
Dicloxacillin sodium, 4-thia-1-azabicyclo [3.2.0] heptane-2-carboxylic acid, 6-[[[3-(2,6-dichlorophenyl)-5-methyl-4-isoxazolyl] carbonyl] amino]-3,3-dimethyl-7-oxo-, monosodium salt, monohydrate is oral penicillinase resistant penicillin. It has isoxazolyl side chain that protects the b-lactum ring from the attack of staphylococcal penicillinase1. It is official in USP (2007) 2.
There are several investigations concerning the determination of Cefixime Trihydrate (CEF) alone and in combination with other drugs in pharmaceutical preparations and in plasma by UV, HPLC, LC-MS, HPTLC methods[3-8] and Dicloxacillin sodium (DICLO) alone and in combination with other drugs in pharmaceutical preparations and in plasma by UV, HPLC, LC-MS, HPTLC methods[9-11]. No references have been found for simultaneous quantitative determination of CEF and DICLO in pharmaceutical preparations. Hence attempts were made to develop simultaneous HPTLC method for the said combination.
In this paper we report simple, accurate, precise and sensitive reverse phase high performance thin layer chromatography method for simultaneous determination of Cefixime trihydrate and Dicloxacillin sodium in combined tablet dosage form. The proposed method is optimized and validated according to ICH guidelines12.
MATERIAL AND METHODS:
Drugs, Reagents and Chemicals used
CEF and DICLO were kindly provided by Maxim Phramaceuticals, Pune, India as gift samples and toluene, triethylamine, Methanol (all AR grade) were purchased from Sisco Research Laboratories Ltd, Mumbai.
Instrumentation
Chromatographic separation was performed on a Merck TLC plates precoated with silica gel 60 F254 (10 cm ×10 cm with 250 mm thickness, E. Merck, Darmstadt, Germany, purchased by Anchrom Technologies, Mumbai, India). The samples were applied onto the plates using Camag 100 microlitre sample (Hamilton, Bonaduz, Switzerland) syringe as a band with 4 mm width using a Camag Linomat 5 applicator (Camag, Muttenz, Switzerland). Linear ascending development was carried out in a twin trough glass chamber (20cm x 10 cm, 10 x 10 cm). Densitometric scanning was performed on Camag TLC scanner 3 at 229 nm for all measurements and operated by winCATS software (V 1.4.2, Camag).
Preparation of Standard Stock Solution
10mg of each of CEF and DICLO were weighed separately and dissolved in 5 mL of AR grade methanol and then volume was made up to 10 ml so as to get the concentration 1000 µg/mL.
Selection of analytical wavelength
From the standard stock solution further dilutions were done using mobile phase and each was scanned in the range of 200- 400 nm and the spectra were overlain. It was observed that both drugs showed considerable absorbance at 229nm as shown in Fig.1 and therefore 229nm was selected as detection wavelength.
229
Fig.1 Overlain Spectra of Cefixime
Trihydrate (CEF) and Dicloxacillin sodium (DICLO)
Preparation of calibration curves
The standard solution of CEF was prepared by dilution of the standard stock solution with methanol to get a concentration of 80µg/mL. From standard solution 1,2,4,6 and 8mL respectively were withdrawn and diluted up to 10mL. 25µL of each of the dilution was spotted on the TLC plate to obtain final concentration of 200, 400, 800, 1200, 1600 ng/band for CEF.
The standard solution of DICLO was prepared by dilution of the standard stock solution with methanol to get a concentration of 200µg/mL. From standard solution 1,2, 4,6,and 8mL were withdrawn and diluted upto 10mL. 25µL of each of the dilution was spotted on the TLC plate to obtain final concentration of 500, 1000,2000,3000,4000 ng/band for DICLO.
The plate was developed in ascending vertical manner using solvent system Tolune : methanol : Triethylamine (4:6:0.7) (v/v/v/v) after 20 min of chamber saturation. Linear ascending development was carried out in a twin trough glass chamber (20cm x 10 cm, 10 x 10 cm) by running the plate at 90 mm. The developed plates were dried and densitometric scanning was performed in the absorbance mode at 229 nm. The slit dimension was kept at 3 x 0.45 mm. After completion of chromatographic analysis, peak areas of both drugs were noted and plotted against corresponding concentrations and least square regression analysis was performed to generate the calibration equation.
Analysis of Tablet formulation
Composition: Each modified release tablet contains
Cefixime Trihydrate IP equivalent to Cefixime 200mg
Dicloxacillin sodium. BP equivalent to Dicloxacillin 500mg
20 tablets were weighed and crushed. A quantity of powder equivalent to 50 mg of CEF was weighed and transferred to a 50 ml volumetric flask. Methanol was added to the same flask and sonicated for 3 to 4 minutes. The volume was made up to 50 ml with methanol. The solution was filtered using whatmann filter paper No.41. The stock solution was diluted suitably to get 16 mcg/ml of CEF and 40mcg/ml of DICLO and spotted 25µL with the help of applicator to get final concentration of 1000ng/band for CEF and 4000ng/band for DICLO. The solutions were spotted keeping 10mm distance between bands. The amount of each drug present per tablet was estimated from the respective calibration curves.
Method Validation12
As per ICH guidelines, method validation parameters checked were specificity, linearity, accuracy, precision, limit of detection, limit of quantitation and robustness.
Specificity
The specificity of the method was ascertained by comparing Rf values and spectra of Standard and sample. The specificity of the method was ascertained by peak purity studies. Purity of the drug peaks was ascertained by analyzing the spectrum at peak start, max position and at peak end. The peak purity was determined by Win CATS software.
Linearity
Linearity of the method was studied by spotting five concentrations of each drug prepared in the methanol, in the range of 200-1600 ng/band for CEF and 500-4000ng/band for DICLO and noting the peak areas.
Accuracy
For accuracy of method standard addition method was followed. It was carried out by applying the method to drug sample to which known amount of both drugs were added separately at level of 80, 100 and 120% of label claim. At each level of the amount, three determinations were performed and the results obtained were compared with expected results.
Precision
The precision of the method was demonstrated by system precision and repeatability.
In System precision 6 replicates of standard solutions of both drugs were made and percentage RSD of the response was calculated. Repeatability was demonstrated by intra-day and inter-day variation studies. In the intra- day studies, 3 repeated measurements were made in a day and percentage RSD of the response was calculated. In the inter day variation studies, 3 repeated measurements were made on 3 consecutive days and percentage RSD of the response was calculated.
Limit of Detection (LOD) and Limit of Quantification (LOQ)
LOD was calculated using the following formula
3.3 x Standard Deviation of the response
LOD = ____________________________________
Slope of calibration curve
LOQ was calculated using the following formula
10 x Standard Deviation of the response
LOQ = ____________________________________
Slope of calibration curve.
Robustness
Robustness of the method was determined by carrying out the analysis under conditions during which time of spotting to development, time of development to scanning were altered and change in wavelength +_2nm from selected and % RSD of the response was determined.
RESULT AND DISCUSSION:
Optimization of Solvent System and Chromatographic Conditions
Chromatographic separation studies were carried out on the stock solution of CEF and DICLO. Initially the plates were spotted with 10µL of stock solution and developed by linear ascending development method using solvents like toluene, hexane, methanol, chloroform, dichloromethane, ethyl acetate, acetone, acetonitrile, etc. Based on the results of these initial chromatograms, binary and ternary mixtures of solvents were tried to achieve optimum resolution and acceptable peak parameter. The final mobile phase consisting of Toluene : Methanol : Triethylamine (4:6:0.7) (v/v/v/v) was selected since optimum resolution and good peaks for both the drugs were obtained as shown in Fig.2. The samples were applied in form of bands of width 4 mm on precoated aluminum sheets of silica gel 60 F254. The application position (X) and (Y) were kept at 10 mm and 10 mm respectively to avoid edge effect. Linear ascending development was carried out in a twin trough glass chamber (20cm x 10 cm, 10 x 10 cm), using 20 mins of chamber saturation. The length of chromatogram run was 90 mm. The plate was dried and scanned at 229 nm over 90 mm distance.
Figure.2 Densitogram of CEF and DICLO
Specificity
Peak purity for the drugs was tested by acquiring spectra at the peak start (S), peak apex (M), and peak end (E) positions. Results from correlation of the spectra were: for CEF r(S, M) = 0.9999 and r(M,E) = 0.9998; for DICLO r(S,M) = 0.9994 and r(M,E) = 0.9994. It can thus be concluded that no impurities or degradation products were found with the peaks obtained from the standard drug solution.
Linearity
When peak area was plotted Vs Concentration (ng/band) both CEF and DICLO showed good linear relationship with correlation coefficient of 0.998 in concentration range of 200–1600 ng/band for CEF and correlation coefficient of 0.997 in the concentration range of 500-4000ng/band for DICLO respectively. Linearity was evaluated by determining five standard working solutions.
The equations of the regression line for CEF was
Y=3.371x+439.5 And that for DICLO
Y=1.816x+821.6
Precision
The proposed method was found to be precise as indicated by percent RSD (% Relative Standard Deviation) of < 1.5 for system precision and repeatability .
Limit of Detection (LOD) and Limit of Quantification (LOQ)
LOD was found to be 7.21ng/band and 9.15ng/band for CEF and DICLO respectively. LOQ was found to be 21.87ng/band and 27.75ng/band for CEF and DICLO respectively.