INTRODUCTION:
Cefadroxil chemically a 7‐[[2‐amino ‐2‐(4‐hydroxyphenyl) acetyl] amino]‐3‐methyl‐ 8‐oxo‐5‐thia‐1‐azabicyclo [4.2.0] oct‐2‐ene‐2‐ carboxylic acid Cefadroxil.1
Chemical structure of Cefadroxil
Cefadroxil, a first‐generation cephalosporin antibiotic, is used to treat urinary tract infections, skin and skin structure infections, pharyngitis and tonsillitis. Like all beta‐lactam antibiotics, cefadroxil binds to specific penicillin‐binding proteins (PBPs) located inside the bacterial cell wall, causing the inhibition of the third and last stage of bacterial cell wall synthesis. Cell lysis is then mediated by bacterial cell wall autolytic enzymes such as autolysins; it is possible that cefadroxil interferes with an autolysin inhibitor. Literature survey revealed that cefadroxil was qualitatively assayed in biological fluids either individually or in presence of other antibacterial drugs using liquid chromatography 5, other new methods and using hydrotope are also there for the determination of cefadroxil 2, 3, 4.
Chemical structure of Probencid
Probenecid is a uricosoric agent used in gout therapy. When Cefadroxil is co-administered with Probenecid, the renal excretion of Cefadroxil is inhibited. The combination is used in gastrointestinal tract and respiratory tract infections. The prototypical uricosuric agent. It inhibits the renal excretion of organic anions and reduces tubular reabsorption of urate. Probenecid has also been used to treat patients with renal impairment, and, because it reduces the renal tubular excretion of other drugs, has been used as an adjunct to antibacterial therapy. [PubChem] .The mechanism by which probenecid inhibits renal tubular transport is not known, but the drug may inhibit transport enzymes that require a source of high energy phosphate bonds and/or nonspecifically interfere with substrate access to protein receptor sites on the kidney tubular.
EXPERIMENTAL:
Pharmaceutically pure samples of CEF and PROB were obtained as gifts from Curex Pharmaceuticals Industries Ltd Jalgaon. Methanol was used as solvent in the study. Double beam UV/Vis spectrophotometer Shimadzu model 1800 with a pair of 10mm matched quartz cells was used to measure absorbance of the resulting solution.
Preparation of standard stock solution:
Accurately 10 mg each of CEF and PROB was weighed separately and transferred to two different 100ml volumetric flask. Volume was made up to the mark with Methanol. The standard stock solutions (100μg/ml) were further diluted separately to obtain working standard of concentration 10μg/ml of CEF and PROB each.
Study of spectra and selection of wavelengths:
Each working standard solution was scanned between the range 200-400 nm in 1 cm cell against blank. Maximum absorbing wavelength of CEF and PROB were selected from spectral data and isobestic wavelength selected from overlain spectra of zero order. The λ max for CEF, PROB and isobestic point was 233nm, 247nm and 242nm respectively.
Fig N0-01
Preparation of Calibration Curve:
From the standard stock solutions six working standards of CEF (10, 20, 30, 40, 50 and 60mg/mL) and six working standard of PROB (10, 20, 30, 40, 50 and 60mg/mL) were prepared. Chromatograms of each solution were recorded for 15 min. The results are shown in Table No.01.The graphs of area Vs concentration were recorded for both the drugs and are shown in Fig. 02and 03. Typical Chromatogram of Standard CEF and PROB are shown in Fig. 04 and 05.
Table No.01: Linearity Study of CEF and PROB
|
Drug |
Linearity Range (µg/mL) |
Slope
|
Intercept |
Corrélation Coefficient (r2) |
|
CEF |
10-60 |
8.1589 |
2.2984 |
0.9985 |
|
PROB |
10-60 |
7.85 |
2.7014 |
0.9984 |
Fig No.2: Calibration Curve of Cefadroxil by using RP-HPLC
Fig No-03: Calibration Curve of PROB by using RP-HPLC
Fig No. 04: Typical Chromatogram of Standard CEF
Fig N0-05:Typical Chromatogram of Standard PROB
Assay:
Twenty micro liters of pure mixed standard solution (n = 6) were injected separately to an injector of HPLC and chromatogram was recorded, from the area amounts of both the drugs were calculated.
Estimation of CEF and PROB in Standard Laboratory Mixture:
In order to check the feasibility of proposed method for simultaneous estimation of CEF and PROB in marketed pharmaceutical formulations, the method was first tried for estimation of drugs in standard laboratory mixture. Accurately weighed CEF(10mg) and PROB (10 mg) were taken in 10 mL volumetric flask, dissolved in mobile phase containg (acetonitril and phosphate buffer, PH 2.5,60:40). Then the content was shaken for 30 min and volume was made up to mark using the same. The above solution was filtered through 0.2m Millipore membrane filter. This solution was further diluted using mobile phase to obtain mixed sample solutions having concentration of CEF (10 μg/mL) and PROB (10 µg/mL).
The amount of each drug estimated in laboratory mixture was calculated using following formula.
Where,
At= AUC for sample solution
As = AUC for standard solution
Ds = Dilution of standard
Dt = Dilution of sample
Ws = Weight of standard (mg)
Wt = Weight of sample (mg)
A = Average weight
Lc = Label claim
The assay results are shown in (Table No. 02) with chromatogram in (Fig.06)
Table No. 02: Assay Result of CEF and PROB in Standard Laboratory Mixture
|
Drug |
Amount present µg/mL) |
Mean area* |
% of Drug found* ± SD |
% RSD |
|
CEF |
10 |
87.0947 |
99.98 ± 0.013 |
0.013 |
|
PROB |
10 |
84.9817 |
99.97 ± 0.016 |
0.016 |
*Average six determinations
Fig N0 .06: Typical Chromatogram of CEF and PROB in Standard Laboratory Mixture
Estimation of CEF and PROB in Marketed Pharmaceutical Formulation:
Twenty tablets were weighed accurately and finely powdered. The powder equivalent to CEF (250 mg) and PROB (250 mg) was accurately weighed and transferred to 100mL volumetric flask containing mobile phase (acetonitril and phosphate buffer, 60:40) (50mL). Then the content was shaken for 30 min. and volume was made up to mark using the same mixture. The above solution was filtered through 0.2m millipore membrane filter. This tablet solution was further diluted with mobile phase to obtain mixed sample solutions having concentration of CEF (10 μg/mL) and PROB (10 μg/mL).
The assay results are shown in (Table No.03, 04) with chromatogram (Fig. 07)
Validation of the Proposed Method:
Accuracy (Recovery Study):
Recovery studies were carried out by adding a known amount of pure drugs CEF and PROB to a preanalysed sample solution. These studies were carried out at 80%, 100% and 120% level. The recovery studies showed that the results were within acceptable limits. The results are given in Table No 05.
Precision:
Precision study was carried out using parameter like method repeatability, intra-day and inter-day precision study by analyzing six replicate standard samples which showed that results were within acceptable limit i.e. RSD below 2.0 indicating that the method is reproducible. The results are shown in Table No. 06, 07 and 08.
Fig N0-07 Typical Chromatogram of CEF and PROB in Marketed Formulation