Amoxicillin 250 mg uses

time curves from 0-8 hours (AUC 0-8h ) were calculated using the trapezoidal method and the first order elimination rate constant (Ke) was estimated using the least square regression of the points describing the terminal log-linear decaying phase. C max and AUC 0-8h data were analyzed statistically using both parametric (one-way ANOVA) and non-parametric methods (Wilcoxon's signed ranks test). The alternative HPLC-UV method described and used here for drug quantification provides the appropriated sensitivity, specificity and high sample throughput required for pharmacokinetic studies.

Figure 2 shows that under described chromatographic conditions, the retention times for AMO and internal standard were 4.2 and 5.2min, respectively.

As also shown in Figure 2, no endogenous interfering peaks appeared at the retention times of the compounds of interest.

Figure 2: Chromatographic analysis of amoxicillin A) plasma blank; B) plasma + internal standard (cefadroxil 15 m g/ml); C) plasma + amoxicillin (20 m g/ml) + internal standard (cefadroxil 15 m g/ml); D) Chromatogram obtained from plasma of a volunteer following oral administration of Amoxicillin (500mg).

The mean absolute recovery of AMO in plasma was 90.0% at 3?g/ml, 98.6% at 25 m g/ml and 95.3 at 50 m g/ml.

The LOD and the LOQ for AMO were 0.1 and 1g/ml, respectively. The calibration curve was linear over the range 1.0g/ml to 50g/ml, with a regression coefficient ? 0.999 and intercept not significantly different from zero (Figure 3). The analytical precision and accuracy obtained for intra-day and inter-day assays of four quality controls (1, 3, 25 and 50 m g/ml; n=5) are shown in Table 1.

Table 1: Analytical precision and accuracy of the determination of amoxicillin from spiked plasma samples (n=5). The overall variability (n=48) was 11.0, 6.5, 5.2 and 4.8% respectively; and the accuracy was 102.8, 103.3, 103.0, 106.8%. No significant degradation of AMO was observed during this period under the storage conditions.

AMO was well tolerated at the administered dose, and no adverse effects were reported. Figure 4 shows mean AMO plasma concentrations as a function of time after the oral administration of 500mg AMO of both brands. The major mean pharmacokinetic parameters derived from the plasma concentration vs.

The geometric mean ratios for AUC0-8h and Cmax of the two AMO oral formulations are shown in Table 3, along with analyses of their position in relation to the 80-125% interval using different tests.

Figure 4: Curves of the mean plasma concentration of amoxicillin (± SEM) of 24 volunteers vs. Table 2: Mean pharmacokinetic parameters obtained in 24 healthy volunteers after the administration of both 500mg amoxicillin formulations.

Table 3: Statistical analysis of AUC 0-8h and C max ratios between Amoxicilina (Test) and Amoxil® (Standard). AMO plasma levels have been measured by other methods using HPLC in combination with UV detection (

14-19

The lowest limit of quantification obtained by UV detection was 50ng/ml, but the elution times for both AMO and internal standard were 31.8 and 32.8 min, respectively, much longer than those reported herein (14). The method of Chulavatnatol & Charles was described only for the measurement of amoxicillin in urine (19). A simple high-performance liquid chromatographic method involving protein precipitation and ultraviolet detection at 220 nm was reported by Hoizey et al. for the simultaneous determination of AMO and clavulanic acid in human plasma (15). Unfortunately, the LOQ obtained for AMO was 635 m g/ml, even higher than in our method (1 m g/ml). Other more complicated extraction procedures such as solid-phase extraction have also been reported (17, 18, 24-26). Nevertheless, solid-phase extraction procedures are laborious and require SPE cartridges, increasing the cost of the analysis. In order to improve sensitivity, a column switching ion-pair reversed phase liquid chromatographic system with a post column derivatization method has been used by Carlqvist & Westerlund (23).

However, their assay, where the limit of quantification was 10ng/ml, has been carried out with no internal standard. In addition the method was complex due to the post column derivatization step.

Their retention time was also longer for the analyte (approximately 10 min as compared to our 4 min) and additionally was not used for a pharmacokinetic study in humans where a large number of samples were analyzed.

Derivatization of AMO to a fluorescent compound showed similar sensitivity and slightly longer retention time (4.5 min for

AMO

and Mascher & Kikuta have satisfactory limits of quantification but do not utilize an internal standard, which is required for an HPLC method meeting GLP guidelines (20, 22).

reported the determination of AMO using LC-MS-MS detection. Mass spectrometers, however, are expensive and not readily available.

Furthermore, the solid-phase extraction procedure employed need laborious sample pre-treatment and requires SPE cartridges, increasing the cost of the analysis (26). The method presented here is less sensitive than many other available assays, i.e., 1 m g/ml.

However, this level of sensitivity was sufficient to perform the bioequivalence study.

The pharmacokinetic study was done function of amoxicillin in 8 hours and the resulting value of AUC 0- Ґ? was less than 20% higher than that of AUC 0-8 for both formulations (12 and 13.9% for Amoxicilina and Amoxil respectively), as recommended by the regulatory agency. Later samples (12 and 24h after the administration of the drug) were taken and analyzed, but no better estimation of AUC 0- Ґ? was observed and the differences between the values remain virtually the same.

The choice of Cefadroxil as an internal standard for AMO was based on the presence of similar functional groups in both structures in addition to their similarity in terms of elemental compositions and chemical behavior. Several wavelengths (225-330nm) were evaluated and 229 nm produced the best results in terms of selectivity and sensitivity.

Considering the fact that the present method involves a shorter running time and a simple sample preparation process, it may be used in similar studies as a time and cost effective alternative to other available methods. The validated HPLC method employed here proved to be simple, fast, reliable, selective and sensitive enough to be used in clinical pharmacokinetic studies of AMO in humans.

The median T max and C max values for both oral formulations were similar to those reported in the literature.

The present study shows that Amoxicilina 500mg capsules was bioequivalent to Amoxil® capsules 500mg, in terms of both the rate and extent of absorption, as assessed by the appearance of 90% CI for both AUC 0-8h and C max ratios within the 80-125% interval proposed by the Food and Drug Administration .

Tomas, A., From penicillin-binding proteins to the lysis and death of bacteria. Tomas, A., The mechanism of irreversible antimicrobial effect of penicillins: how the beta lactam antibiotics kill and lyse bacteria.

L., Interaction of penicillin with the bacterial cell: penicillin-binding proteins and penicillin-sensitive enzymes.