Amoxicillin kidney pain

In such cases, substitution of large volume tablet with two capsules containing the same drug strength can solve problems associated with swallow ability. However, bioequivalence problems may arise amoxicillin for inflammation especially when the substitution was carried out between liquid and solid oral dosage forms, or between soft elastic gelatin capsules and tablets[4].

On the other hand, bioequivalence cannot be compromised when two solid dosage forms such as hard gelatin capsules and tablets where interchanged. Few publications are found in literature demonstrating potential interchangeability of two different solid dosage forms based on pharmacokinetic data[5,6]. The aim of the present study was to determine if the practice of interchangeability between two amoxicillin capsules (Amoxil ® , 500 mg/capsule) and one amoxicillin tablet (Amoxicare ® , 1000 mg/tablet) in order to achieve better patient compliance can be conducted without compromising their extent of absorption.

Acetonitrile HPLC grade, methanol HPLC grade and tetra-n-butyl ammonium hydroxide were from Merck, (Darmstadt, Germany); chloroform HPLC grade was from Fisons Scientific Equipments, UK; sodium dihydrogen phosphate dihydrate (NaH 2 PO 4 ·2H 2 O) was from Riedel de Haen, (Seelze, Germany); double distilled high

purity

Amoxicillin and Paracetamol (internal standard) were supplied by Pharmacare Chemical and Cosmetics (Ramallah, Palestine). Amoxicare ® tablet (1000 mg amoxicillin/tablet, batch number RD-23E03) was from Pharmacare Chemical and Cosmetics. Amoxil ® capsules (500 mg/capsule, batch number 040605) were manufactured by Medical Union Pharmaceuticals Co, Abou Sultan Egypt, under license of Beecham Pharmaceuticals Brantford England. The protocol of the study was approved by the Ethical Committee of Tanta University Hospital (Tanta, Egypt). The whole study followed the requirements of the Declarations of Helsinki and was conducted in accordance with the current Good Clinical Practice (GCP), International Conference Harmonization (ICH) as well as Good Laboratory Practice (GLP) Guidelines.

Each volunteer had signed the informed consent form after ample information and consideration time has been provided. Twenty-four adult male volunteers, non-smokers, aged between 17-30 y, weighing between 59 and 85 kg, were chosen to participate in the present study.

The volunteers were not on concomitant medications and they 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 and physical examinations. Subject’s health status was determined following a physical examination, laboratory tests and medical history by a qualified registered MD physician. The physician reviewed all preclinical laboratory tests for each subject. Exclusion criteria included extreme weight ranges (overweight or underweight), anemia, liver or renal dysfunction, parasitic and other diseases or conditions that was judged to affect the absorption, distribution and/or elimination of amoxicillin. The subjects were asked to abstain from taking drugs and alcohol for at least 3 days prior to the test and throughout the entire study period.

However, it has to be underlined that the drugs eventually used by the involved volunteers are characterized by short half life values and thus do not overlap with amoxicillin during the first day of study.

On the night before starting the study, the volunteers were instructed to fast for at least 10 h before drug administration.

The study had an open randomized two-period crossover design with a 7-day washout period between doses.

The volunteers were randomly divided into two equal groups each of 12 subjects. The first group was given the reference formulation and the second group received the test formulation with a crossover after a washout period of one week. On the morning of the experiment, 10 ml of blood sample was withdrawn from each volunteer to serve as a blank for the drug assay.

Each of the 24 volunteers then took one Amoxicare ® tablet (1000 mg amoxicillin) as the test drug or two Amoxil ® capsules (500 mg amoxicillin each) as the reference drug followed by 200 ml of water. Five milliliters blood samples for plasma drug assay were collected from an indwelling catheter inserted in the antecubital vein of one of the arms. Samples were obtained at 0.0 (blank sample), 0.25, 0.5, 0.75, 1, 1.5, 2, 2.5, 3, 4, 6, 8 h after drug administration, in heparinized tubes.

Plasma was directly separated by centrifugation at 3000 rpm for 10 min and stored at -20° until analyzed.

Four hours after drug administration, the subjects were allowed to eat a standard breakfast of bread, jam, low-fat white cheese, water (150 ml).

They were then allowed controlled access to fruit juice and other non-alcoholic beverages. The volunteers had their second meal (standard lunch containing grilled chicken, rice and vegetables) 4 h later.

Stock solution of amoxicillin (500 µg/ml) was prepared by dissolving 50 mg of drug in methanol. Working standard solutions were prepared from the stock solution by serial dilution with methanol to prepare seven working solutions of amoxicillin with concentrations of 5, 10, 20, 50, 100, 200 and 500 µg/ml. Stock solution of paracetamol (internal standard) was prepared by dissolving the drug powder in methanol to give 50 µg/ml.

Stock and working standard solutions were protected from light and stored at -20° until use.

The calibration standards were prepared by adding known amounts (50 µl) of amoxicillin to each working solution, and 50 µl of the internal standard solution to a set of clean test tubes.

After evaporation of the methanol solution 0.5 ml of blank plasma was added to each tube to form a set of calibration standards with concentrations of 0.5, 1.0, 2.0, 5.0, 10, 20 and 50 µg/ml. The calibration standards were treated with 1 ml of acetonitrile and shaken for 1 min, then centrifuged at 3000 rpm for 7 min.

The supernatant was transfered to clean test tube and 5 ml of chloroform was added, shacked for one minute, and then centrifuged at 3000 rpm for 7 min.

The upper layer was separated and 50 µl was injected into the HPLC column. For the analysis of the samples, 0.5 ml of each study sample was transferred to a clean test tube spiked with the internal standard.

The study samples were treated as the calibration standards after addition of the internal standard.

Under the chromatographic conditions amoxicillin for wounds described above, the retention time of amoxicillin and the internal standard (paracetamol) were 4.8±0.5 and 6.0±0.5 min, respectively. Analytical validation report for HPLC method used for the quantification of amoxicillin in human plasma is described below.

The analyses were performed using an HPLC system equipped with a variable wavelength UV detector and an automatic injector was used in this study Waters 2690 Separation Modules, Waters Corporation, Milford Massachusetts, USA. Separation was accomplished with a 250?4.6 mm 5 µm Hypersil ® BDS C 18 column, Thermo Fisher Scientific Inc, Waltham, United States.

The mobile phase used consists of 94% buffer and 6% acetonitrile, where buffer is a mixture of tetra-n-butyl ammonium hydroxide (0.15 g/l) in sodium dihydrogen phosphate dehydrate (20 mM). The flow rate was 1.2 ml/min, the UV detector was set at 225 nm and the peak areas were calculated using the Millennium data analysis program (Millennium Consultants Inc, NJ, USA). Quantification of amoxicillin was obtained by plotting amoxicillin to internal standard peak area ratios as a function of concentration.

For statistical analysis a Minitab statistical Package version 13 on IBM PC was used.

The validation of this chromatographic analytical method was performed in order to evaluate its linearity, selectivity, stability, precision and accuracy. Calibration curves were constructed from the peak area ratio (drug/ internal standard) and the corresponding amoxicillin concentration in each calibration standard.

The linearity study was carried out in the range of concentrations between 0.5 to 50 µg/ml.

To assess linearity, drug free plasma was spiked

with

The correlation coefficient was always greater than 0.99 during the course of the validation. METHOD VALIDATION PARAMETERS OF AMOXICILLIN ANALYSIS. Parameters Results Linearity range 0.5-50 mg/ml Correlation coefficient 0.9978 Regression equation (y=mx+q) m 0.208 c 0.056 Limit of quantitation (LOQ) 0.5 µg/ml Coefficient of variation intraday (%CVintra) (n=3) from 0.735 to 13.38% Coefficient of variation inter-day (% CVinter) (n=3) from 0.783 to 13.13%

Accuracy

109%

Precision and accuracy data were obtained by analyzing aliquots of three-spiked plasma at low middle and high concentration levels of amoxicillin. Intraday reproducibility was determined by analyzing three replicates of calibration curves on the same day, and inter-day reproducibility was evaluated by the analysis of six different calibration curves on six different days during the study period.

The intraday coefficient of variation and the inter-day coefficient of variation were within 10% indicating that the method is precise.

The accuracy of the method was validated as the intra-day accuracy was in the range from 95 to 109% and the inter-day accuracy was in the range of 93 to 109% during the entire period in which calibration curves were generated. The drug stability at room temperature was determined preparing three different plasma samples of each drug concentration and then injecting the samples immediately into the HPLC system.

The samples were kept at room temperature and were injected again after 8 h.

The concentrations measured at time zero and after 8 h were compared to determine whether there were changes in the concentrations with time. The difference in the drug concentrations in the two analyses was always less than 10%.

When the samples were kept at room temperature for 8 h, no changes in the drug concentration was evident thus indicating the stability of amoxicillin in the plasma samples at room temperature.

The stability of the drug in frozen plasma was investigated by the analysis of samples obtained from three volunteers. The first analysis was performed at the beginning of the study and the second analysis was performed at the end of the study.

The samples were stored at -20° C between the analyses.

The difference in the drug concentration in all samples in the two analyses was always less than 10% in each sample.

There was no change in the drug concentration in frozen plasma during storage at -20° during the study period, indicating stability of amoxicillin in frozen plasma. The lower limit of quantification (LLOQ) was estimated by analyzing samples with known amounts of amoxicillin, at progressively lower concentrations, starting at the lower end of the calibration curves. The limit of quantification of amoxicillin in this assay was 0.5 µg/ml. The retention time of the drug in the standard and the study samples were identical.

There were no peaks for endogenous compounds that appeared at the same retention time for amoxicillin in the chromatograms for six different blank plasma samples.

The concentration of amoxicillin in the plasma (µg/ml) at different time points following administration of either one Amoxicare ® tablet (1000 mg amoxicillin) or two Amoxil ® (500 mg amoxicillin each) were plotted for each volunteer.

The area under the curve of plasma drug concentration (AUC) versus time (t), from zero to t (AUC 0>t ), was calculated from the drug concentration by the linear trapezoidal rule, using the relationship; where, C n is the drug concentration at any time (t n ).

The area under the curve from t h to infinity was obtained from the relationship: where, C t is the concentration of amoxicillin at the t-hour time point (last determined concentration), K e is the elimination rate constant in a particular individual. The elimination rate constant (K e ) was calculated from the slope of the straight part of log C versus t plot, where the slope of the straight line equals (-k/2.303). To obtain this value, the data were plotted as log plasma concentration versus time (to ensure linearity of the terminal phase of the profile) and the best fitting line was determined by the method of least square.

The total area under the curve from zero to infinity (AUC 0>? ) was calculated as the sum of the areas obtained. Following the oral administration of drugs, the plasma concentration generally reaches a single, well-defined peak (C max ) at the time of T max .

C max is an important kinetic index for drug safety and can be viewed as a measure of maximal exposure. Particularly, C max has important roles in clinical pharmacological investigations, including bioequivalence, T max has been considered as a simple measure for comparative absorption and disposition rate constants.

C max and T max can be estimated either directly or indirectly.

In the first case, one can record the maximal observed concentration and the corresponding time. In the second case, a pharmacokinetic model can be fitted to the measurements and the predicted maximum evaluated[7].

The maximum amoxicillin plasma concentration (C max ) and the corresponding time of peak plasma concentration (T max ) were taken directly from the slope of the semi-logarithmic plot of the terminal phase of the plasma concentration-time curve calculated by linear

regression

For the purpose of bioequivalence analysis, one-way analysis of variance (ANOVA procedure) was used to assess the effect of formulations, periods, sequences and subjects on AUC 0-8 , AUC 0-? , and C max .

The statistical analysis was performed using Minitab Statistical package version 13 IBM PC (Minitab Inc.

The alternate HPLC-UV method described and used here for amoxicillin quantification provided the required sensitivity, specificity and high sample throughput required for pharmacokinetic studies.

2 are shown three representative chromatograms of amoxicillin. Particularly, blank plasma sample chromatogram, blank plasma spiked with amoxicillin (20 µg/ml) chromatogram and plasma sample of volunteer #14R obtained after 1.5 h from administration, are reported.

Both amoxicillin formulations (one Amoxicare ®

1000

All volunteers were discharged from the hospital in good health conditions. Both medications were quantifiable at the first sampling time in all the volunteers.