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HPLC DETERMINATION OF BIO-AVAILABILITY OF AMOXICILLIN CHITRALEKHA SAINI* Ph.D.Scholar, Research Lab. Department of Botany, Dungar College, Bikaner (Raj) ABSTRACT An accurate precise and sensitive HPLC assay was developed for determination of amoxicillin in human plasma samples, to compare the bioavailability of two amoxicillin capsule 500mg formulation (Amoxicillin from Brazil. as a test formulation and Amoxil from smith Kline Beecham Laboratories Brazil, as a reference formulations) in 24 volunteers both sexes. KEY WORDS: Amoxicllin (AMO), HPLC Human Plasma INTRODUCTION Amoxicillin [{ 2S-[2,5,6 (5*)}]-6-{[Amino (4-hydroxyphenylacetyl]amino}-3,3-Dimethyl-7-oxo4-thia-1-azabicylo[3,2,0]heptane-2-carboxilic acid] is an oral semi-synthetic penicillin structurally related to ampicillin.

Figure - 1 Amoxicillin Amoxicillin (AMO) show high absorption after oral administration and this is not altered by the concomitant ingestion with food. AMO reaches Cmax (8g/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 I hour, 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. AMO is commercially available is the form of capsules and Tablets containing 250 and 500 mg ( as amoxicillin free base) for oral administration .It is also available in the form of suspensions containing 125 and 250 mg/5ml. AMO is presently the most commonly used antibiotic. To understand the pharmacokinetic behavior of AMO in human 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 (= 225-330nm). fluorimetric detection paired reagents and special techniques such a post colomn derivatization or column switching 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, liquid- liquid extraction or more complicated extraction such as solid- phase extraction. The purpose of this paper is 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 Beccham. After evaluation of various conditions of the HPLC assays, a suitable and simple assay for the measurement of amoxicillin in human plasma was developed using reversed- Phase HPLC and direct UV detection. MATERIAL AND METHODS DRUG AND CHEMICALS acetonitrile (ACN) methanol (CH3OH) Sodium hydroxide (NaoH) and sodium phosphate salts (Na2 HPO4 / NaH2 PO4)
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The HPLC grade solvents ACN and CH3OH were used as received; all other reagents were analytical grade Amoxicillin and Cefadroxil (internal standard). The deionized water was prepared using milliQ- system. STANDARD SOLUTIONS Stock Solution of AMO (1 mg/ml) was prepared in H2o: ACN (95:5) working standard solution was prepared from the stock solution by sequential dilution with H2o: ACN (95:5) to yield final concentration of 1, 10 and 100 g/ml. Stock solution of Cefadroxil (internal standard) was prepared in CH3OH (1mg/ml) stock and working standard solutions were protected from light and stored at -20oC until used. calibration standard were obtained by adding known amount of AMO to drug free plasma to achieve the concentrations of 1,5,10,20,40 and 50g/ml. Three quality controls of low (3g/ml) middle (25g /ml) and high (50 g/ml) concentrations were prepared by adding known amount of AMO to drug free plasma. Plasma solutions were protected from light and stored at -70oc until used. EXTRACTION PROCEDURE AMO and cefadroxil (internal standard) were extracted from human plasma samples by protein precipitation. A200 aliquos of each plasma sample was transferred to a 1.5ml polypropylene tube. Then 15ul of 1mg/ml internal standard solution and 400 l of cold CH3OH ( kept on ice) were added after a brief vortex mixing, the tubes were centrifuged (14000 rpm at 4 OC for 15 min) . A 100 ll aliquot of the supernatant was transferred to the injection vials and 20l were injected in to 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. INSTRUMENTS AND CHROMATOGRAPHIC CONDITIONS The analyses were performed on a shimadzu chromatographic system equipped with a LC- 10 Ad VP pump, an SIL- 10AD VP auto- sampler, an SPD- 10A VP UV detector and on SCL 10A-VP controller unit. the drug analysis data were acquired and processed using CLASS-VP 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 (95.5v/v) pumped at a flow rate of 1.3 ml/min through the column at room temperature peaks were monitored by UV absorbance at 229 nm, Sensitivity of 0.005AUFS. Quantification of AMO was obtained by plotting AMO to internal Standard peak height ratios as a function of concentration. STABILITY Drug free plasma was spiked with known amount of the drug to achieve the concentrations of 3,25, 50g/ml (n=3) and stored at-700C. 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 SPECIFICITY The specificity of the method was determined by comparing the chromatogram obtained from the samples containing AMO and internal standard with those obtained from blank samples. LIMIT OF DETECTION (LOD) and limit of Quantification (LOD) 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 single to noise ratio and comparing test results from samples with known concentration 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% RECOVERY AND LINEARITY
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The analytical recovery of AMO was determined at concentrations of 1,3,25 and 50g/ml (n=3) Drug free plasma was spiked with known amounts of the drug to achieve the concentration previously specified these sample were processed by the analytical method described above and peak heights were compared with the peak hights obtained by direct injection of the drugs in the mobile phase. The linearity study was carried out in the range of 1 to 50g /ml (n=4) to access linearity drug free plasma was spiked with known amount of the drug to achieve the concentrations of 1, 5,10,20,40 and 50 g/ml PRECISION AND ACCURACY 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 (3g/ml) middle( 25g/ml) and high (50g/ml) concentration level 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). FORMULATIONS The following test formulation was employed: 500mg amoxcillina capsule from Brazil (AC 309/ production date 6/2006 Expiry date 6/2008). The details of reference formulation are as follows 500 mg Amoxil Capsules produce by smithkline Beechin (BB0028/Production date 3/2006, Expiry date 3/2008.) CLINICAL PROTOCOL Twenty four (12 male and 12 female) adult volunteers, non smokers, aged between 21 and 41years, weighting between55 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 hemato logical disease as determined within four weeks prior to the beginning of the study by way of medical histories, physical examinations and the following laboratory screening, fasting blood glucose, urea, creatinine, SGOT (AST), SGPT (ALT) total bilirubin , total protein, plasma albumin, alkaline, phosphatasis , sodium, potassium, chlorides, uric acid, urinalysis, hemoglobin, hematocrit and total differential white blood cells count. The study had an open randomized two period crossover design with a 7-day washout period between doses. During each period, volunteers were hospitalized, head 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 200 ml of tap water Blood sample for plasma drug assay were taken from a forearm vein at 0, 0.5, 1.5,2,3,6 and 8 hours after AMO administration on the each occasion one 5ml sample was taken via "butterfly" in to a clen tube. After blood clotting at room temperature, the blood samples were centrifuged at 2000rpm for ten minutes and the plasma removed and stored at-700C until assayed. The volunteers received 200ml of tap water drink three hours after dose administration. Six hours after dose administration a standard lunch was mode available and on evening meal was provided 12 hours after administration of the dose. But xanthine-containing beverages, Such as tea, coffee and cola were not permitted. PHARMACOKINETIC AND STATISTICAL ANALYSIS maximum observed plasma concentration (Cmax) and time taken to reach it (Tmax) were obtained from drug concentration vs. time curves. The areas under the AMO concentrations vs time curves from 0-8 hours (AUC 0-8h) Area under the plasma Concentration-Time curve) 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. T 1/2 were drived from ke (T1/2=In 2/ke). Cmax and AUCo-8h data were analyzed statistically using both parametric (oneway ANOVA) and non-parametric methods (wilcoxons signed ranks test. RESULTS

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Thealternative HPLC- UV method described and used here for drug quantification provides the appropriated sensitivity, specificity and high sample through put required for pharmacokinetic studies. Figure-2 Shows that under described chromatographic conditions, the retention times for AMO and internal standard were 4.2 and 5.2 min, respectively. An also show in figure-2, no endogenous inferfering peaks appeared at the retention time of the am pounds of interest

(A) (B) (C) (D)

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Figure-2 Chromatographic analysis of Amoxicillin Plasma blank Plasma + internal standard cefadroxi 15g/ml) Plasma + amoxicillin (20 g/ml ) +internal standard (cefadroxil 15 g/ml) 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 3g/ml, 98.6% at 25g/ml and 95.3 at 50 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

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Figure-3 Calibration curve of Amoxicillin The analytical precision and accuracy obtained for intra-day and inter-day assays of four quality controls (1, 3, 25 and 50 mg/ml n=5) are show in table-1 Table1: - Analytical precision and accuracy of the determination of amoxicillin from spiked plasma samples (n=5) Concentration Concentration CV RE Added (mg/ml) Obtained (mg/ml) (%) (%) Intra-day 1.0 1.05 7.6 105.1 3.0 3.11 4.0 103.8 25.0 26.55 5.0 106.2 40.0 43.68 0.6 109.2 Inter-day 1.0 1.06 4.8 106.4 3.0 3.05 3.2 101.7 25.0 25.55 3.3 102.2 40.0 41.44 5.0 103.6 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.

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FIGURE-4 Show means AMO plasma concentrations as a function of time after the oral administration of 500mg AMO of both brands. The major mean pharmacokinetic paramenters docived from the plasma concentration v/s time curves are presented in table-2, The geometric mean ratios for AVCO-8h and CMAX of the two AMO oral formulations are show in Table-3, along with analyse 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 volunteer's vs time (h) Table-2 Mean pharmaco kinetic parameter obtained in 24 healty volonteers after the administration of both 500mg amoxicillin for mulations.

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Table-3 Statistical analysis of AUC Amoxil (Standad.)

0-8h

and Cmax

ratios between Amoxicilina (Test) and

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REFERENCES 1. Archana Nadiminti, Ashwini Gunda, 2012. Journal of pharm Res, 5(4) 1889-1995. 2. Blumberg P.M. and strominger J.L.1947 interaction of penicillin with bacterial cell penicillin bincling protein and penicillin sensitive enzyme. Bacterial rev. 38:291. 3. J. Batt, S. singh, G. subhaiah, S. Kambi and S. Ameta, 2007. "A rapid and sensitive liquid chrometography. Tendem mass spectrometry (LS-MS/MS) method for estimation of amlodopine in human plasma", Bioredical chromatography. vol 21 no.2, pp. 169-175. 4. Miyazaki k, ohtanik, sunadak, Aritai, 1983. Determination of ampicillin, amoxicillin cephalexin and cephradine in plasma by high- performance liquid chromatography using fluorometric detection. D.J. Chromatogr, 276:478:82, 5. New, H.C. 1974 Antimicrobial activity and human pharmacology of amoxicillin J infect Dis, 129 (suppl): S 123-S131. 6. Pan RN, KUO BP, Pao LH, 2012. validated LC-MS-MS method for the determination of Quetiapine in human plasma Application to a Pharmacokinetic study Journal of chrometography science: SO: 277-82. 7. Pandey s. Pandey p.Ttiwari G & Tiwari R, 2010. Bioanalysis in drug discovery and development pharmaceutical method 1(1) 14-24. 8. Waxman, D.J. and strominger J.L.1983 penicillin- binding proteins and the mechanisms of action of beta- loctam antibiotis. Annu Rev Biochem, 52:825. 9. Yuan , z ; Russlie, H, Q; Canafax, D.M.1995 Sensitive assay for measuring amoxicillin in human plasma and middle ear fluid using solid phase extraction and reversed phase highperformapce liquid chromatography J chromatogr B. 674;93-9

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