Amoxicillin for dental abscess

The buy amoxicillin for human online assay showed excellent relationships between peak height ratios and plasma concentrations (r 2 ? 0.999). The limit of quantification was 1g/ml, based on 200l of plasma.

The geometric mean of Amoxicilina/Amoxil® 500mg capsules individual percentage ratio was 101.4% for AUC0-8h, and 99.9% for Cmax.

The 90% confidence intervals were 98.3-104.4% and 95.7-103.9%, respectively. CONCLUSION : This simple, rapid and selective method is suitable for pharmacokinetic, bioavailability and bioequivalence studies. Since the 90% CI for both C max and AUC 0-8h lies within the 80-125% interval proposed by the Food and Drug Administration, it was concluded that Amoxicilina 500mg capsules was bioequivalent to Amoxil® capsules 500mg, in terms of both the rate and extent of absorption. Amoxicillin [[2S-[2 a ,5 a ,6?(S*)]]-6-[[Amino(4-hydroxyphenyl)acetyl]amino]-3,3-dimethyl-7-oxo-4-thia-1-azabicyclo[3.2.0]heptane-2-carboxilic acid] is an oral semi-synthetic penicillin structurally related to ampicillin (Figure 1).

The presence of a benzyl ring in the side chain extends the antibacterial activity to gram-negative bacteria (1-4).

The action mechanism of these antibiotics has not been unequivocally established, but it is thought they may interfere with peptidoglycan bacterial cell wall synthesis in the effected organisms (5-10). Figure 1: Structures of ampicillin, amoxicillin and cefadroxil (internal standard).

Amoxicillin (AMO) shows high absorption

after

The elimination of the drug occurs preferentially by excretion in the urine, with about 60% of an orally administered dose and 75% of a parenteral dose being excreted unchanged (11-13). AMO is commercially available in the form of capsules and tablets containing 250 and 500mg (as amoxicillin free base) for oral administration.

It is also available in the form of suspensions containing 125 and 250mg/5ml.

AMO is presently the most commonly used antibiotic.To understand the pharmacokinetic behavior of AMO in humans, a reliable quantitative method is needed. Several high performance liquid chromatography (HPLC) methods for the determination of amoxicillin in body fluids have been developed. Most of the methods use direct UV detection at low wavelengths (?=225-330 nm) (14-19). Fluorimetric detection (20-22), ion-paired reagents and special techniques such as postcolumn derivatization or column switching (14, 20, 23) have been used to enhance sensitivity and selectivity. Different methods of sample preparation have been applied prior to the chromatographic analysis, mostly based on protein precipitation (15, 19, 20), liquid-liquid extraction (21) or more complicated extractions such as solid-phase extraction (17, 18, 24-26). The objective of this study was to compare the pharmacokinetic profiles and to evaluate the bioequivalence of two AMO formulations, in 24 healthy volunteers of both sexes. The test AMO capsule (500mg) formulation from Brazil was compared with a commercial AMO capsule (500mg) formulation produced by SmithKline Beecham, which has been defined as the reference standard by the national regulatory agency (ANVISA).

After evaluation of the various conditions of the HPLC assay, a suitable and simple assay for the measurement of amoxicillin in human plasma was developed using reversed-phase HPLC and direct UV detection.

Acetonitrile (ACN) was purchased from EM Science (Gibbstown, NJ, USA); methanol (MeOH), sodium hydroxide (NaOH) and sodium phosphate salts (Na 2 HPO 4 /NaH 2 PO 4 ) were purchased from Merck (Rio de Janeiro, RJ, Brazil). The HPLC grade solvents

ACN

Amoxicillin and cefadroxil (internal standard) were purchased from USP Pharmacopeia (New York, NY, USA). The deionized water was prepared using Milli-Q system (Millipore, Molsheim, France). Stock solution of AMO (1mg/ml) was prepared in H 2 O:ACN (95:5).

Working standard solutions were prepared from the stock solution by sequential dilution with H 2 O:ACN (95:5) to yield final concentrations of 1, 10 and 100 ?g/ml. Stock solution of cefadroxil (internal standard) was prepared in MeOH (1mg/ml). Stock and working standard solutions were protected from

light

Calibration standards were obtained by adding known amounts of AMO to drug-free plasma to achieve the concentrations of 1, 5, 10, 20, 40 and 50 m g/ml. Three quality controls of low (3?g/ml), middle (25 m g/ml) and high (50 m g/ml) concentrations were prepared by adding known amounts of AMO to drug-free plasma.

Plasma solutions were protected from light and stored at -70°C until used. AMO and cefadroxil (internal standard) were extracted from human plasma samples by protein precipitation. A 200 m aliquot of each plasma sample was transferred to a 1.5 ml polypropylene tube. Then, 15 m l of 1 mg/ml internal standard solution and 400 m l of cold MeOH (kept on ice) were added.

After a brief vortex mixing, the tubes were centrifuged (14000 rpm at 4°C for 15 min). A 100 m aliquot of the supernatant was transferred to the injection vials and 20 m l were injected into the chromatographic system.

Quality controls were performed in duplicate for each batch.

All samples from a single volunteer were analyzed on the same day in order to avoid inter-assay variation.

The analyses were performed on a Shimadzu chromatographic system (Kyoto, Japan) equipped with a LC-10AD VP pump, an SIL-10A VP auto-sampler, an SPD-10A VP UV detector and an SCL 10 A-VP controller unit. The drug analysis data were acquired and processed using CLASS-VP (v.6.2) software running under Windows 98 on a Pentium PC. The mobile phase involved a mixture of phosphate buffer (0.01mol/L), pH=4.8 and ACN (95:5 v/v), pumped at a flow rate of 1.3ml/min through the column (Lichrosorb® 10 m m RP 18, 250 x 4.6mm with a guard column Securityguard® C18 10 m m, 4 x 3.0mm; Phenomenex, CA, USA) at room temperature.

Peaks were monitored by UV absorbance at 229nm, sensitivity of 0.005AUFS.

Quantification of AMO was obtained by plotting AMO to internal standard peak height ratios as a function of concentration. Drug-free plasma was spiked with known amounts of the drug to achieve the concentrations of 3, 25 and 50 m g/ml (n=3) and stored at -70°C.

Those samples were used to investigate the stability of AMO over a period of 1 month. No internal standard was added prior to the analysis. The specificity of the method was determined by comparing the chromatograms obtained from the samples containing AMO and internal standard with those obtained from blank samples. Limit of detection (LOD) and limit of quantification (LOQ) LOD is a parameter that provides the lowest concentration of analyte in a sample that

can

The analyte concentration that produced a signal-to-noise ratio of 3:1 was accepted as the LOD. The LOQ is defined as the lowest concentration of analyte that can be determined with acceptable precision and accuracy under the stated experimental conditions. The LOQ was estimated by analyzing samples with known amounts of AMO, at progressively lower concentrations, starting at the lower end of the calibration

curves

precision

The analytical recovery of AMO was determined at concentrations of 1, 3, 25 and 50 m g/ml (n=3). Drug-free plasma was spiked with known amounts of the drug to achieve the concentration previously specified. These samples were processed by the analytical method described above and peak heights were compared with the peak heights obtained by direct injection of the drugs in the mobile phase.

The linearity study was carried out in the range of 1 to 50 m g/ml (n=4). To access linearity, drug-free plasma was spiked with known amounts of the drug to achieve the concentrations of 1, 5, 10, 20, 40 and 50 m g/ml.

Precision was determined as the coefficient of variation (CV), and the accuracy as the percentage relative error (RE). Precision and accuracy data were obtained by analyzing aliquots of three spiked plasma at low (3 m g/ml) middle (25 m g/ml) and high (50 m g/ml) concentration levels of AMO.

Intra-day reproducibility was determined by analyzing 5 aliquots of spiked human plasma and inter-day reproducibility was determined over a 5-day period (n=5). The following test formulation was employed: 500mg Amoxicilina capsules from Brazil (AC 309/Production date 03/2000; Expiry date 03/2002).

The details of the reference formulation are as follows: 500mg Amoxil® capsules produced by SmithKline Beecham (BB0028/Production date 01/2000; Expiry date 01/2002).

Twenty-four (12 male and 12 female)

adult

All volunteers gave written informed consent and the Ethics Committee of Sao Francisco University Hospital approved the clinical protocol. The study had an open randomized two-period crossover design with a 7-day washout period between doses.

During each period, volunteers were hospitalized, had a regular meal, and received a 500mg capsule of the AMO allocated according to the appropriate dose randomization code. After administration of the capsule, the volunteers were asked to drink 200ml of tap water.

Blood samples for plasma drug assay were taken from a forearm vein at 0, 0.5, 1, 1.5, 2, 3, 4, 6 and 8 hours after AMO administration.

On each occasion, one 5ml sample was taken via "butterfly" into a clean tube. After blood clotting at room temperature, the blood samples were centrifuged at 2000g for ten minutes and the plasma removed and stored at -70°C until assayed.

The volunteers received 200ml of tap water drink three hours after dose administration.

Six hours after dose administration a standard lunch was made available and an evening meal was provided twelve hours after administration of the dose. Liquids were allowed ad libitum after lunch, but xanthine-containing beverages, such as tea, coffee and cola were not permitted. Maximum observed plasma concentration (C max ) and time taken to reach it (T max ) were obtained from drug concentration vs.

time curves from 0-8 hours (AUC 0-8h ) were calculated using the trapezoidal method and the first order elimination

rate

constant

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