Moxiclav amoxicillin

Received 10 July 2002, Revised 28 April 2003, Accepted 16 June 2003. PURPOSE : An accurate, precise and sensitive HPLC assay was developed for the determination of amoxicillin in human plasma samples, to compare the bioavailability of two amoxicillin capsule (500mg) formulations (Amoxicilina from Brazil, as a test formulation and Amoxil® from SmithKline Beecham Laboratories Ltda., Brazil, as a reference formulations) in 24 volunteers of both sexes. METHODS : Amoxicillin concentrations were analyzed by combined reversed phase liquid chromatography and UV detection (?=229 nm).

Amoxicillin and cefadroxil (internal standard) were extracted from the plasma by addition of cold methanol. The separation was achieved

using

The mobile phase consisted of a 95% phosphate buffer (0.01mol/L), pH=4.8 and 5% acetonitrile mixture. The study was conducted using an open randomized 2-period crossover balanced design with a 1-week washout period between the doses.

Plasma samples were obtained over an 8-hour period.

The bioequivalence between the two formulations was assessed by calculating individual peak plasma concentrations (C max ) and area under the curve (AUC 0-8h ) ratios (test/reference).

The statistical interval proposed was 80-125%, as established by the US Food and drug administration Agency. RESULTS : The internal standard and amoxicillin eluted about 4.2 and 5.2 min, respectively at a flow rate of 1.3ml/min. The mean absolute recovery of AMO in plasma was 90.0% at 3 m g/ml, 98.6% at 25 m g/ml and 95.3 at 50 m g/ml.

The 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

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

has

established

Amoxicillin (AMO) shows high absorption after oral administration, and this is not altered by the concomitant ingestion with food (6-8). AMO reaches C max (8 m g/ml) about 2 hours after administration, exhibits low binding with plasma proteins (17%), is quickly distributed through the body, and has an elimination half-life of 1 hour (7-10). 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

plasma

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 and stored at -20°C until used.

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 be detected, but not quantified, under the stated experimental conditions. The LOD was determined by using the signal-to-noise ratio and comparing test results from samples with known concentrations of analyte against blank samples. 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.

The LOQ was considered as the concentration level in which accuracy and precision were still better than 20%. 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

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

Twenty-four (12 male and 12 female) adult volunteers, non-smokers, aged between 21 and 47 years, weighing between 55 and 95 kg and within 15% of the ideal body weight, were selected for the study.

The volunteers were not on concomitant medications and were free from significant cardiac, hepatic, renal, pulmonary, gastrointestinal, neurological or hematological disease as determined within four weeks prior to the beginning of the study by way of medical histories, physical examinations and the following laboratory screenings: fasting blood glucose, urea, creatinine, SGOT (AST),

SGPT

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

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.