DEVELOPMENT AND VALIDATION OF AMOXICILLIN AND CLAVULANATE BY USING LC-MS METHOD

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1 WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Parijatha et al. SJIF Impact Factor Volume 6, Issue 1, Research Article ISSN DEVELOPMENT AND VALIDATION OF AMOXICILLIN AND CLAVULANATE BY USING LC-MS METHOD B. Parijatha 1*, K. Anitha 2, K. Prashanthi 1, D. Santhoshi Priya 1, K. Durga Prasad 2 and Krishnamohan Chinnala 1 1 School of Pharmacy, Nalla Narasimha Reddy Education Society s Group of Institutions, Hyderabad, Telangana, INDIA. 2 Mother Theresa College of Pharmacy, NFC Nagar, Hyderabad, Telangana, INDIA. Article Received on 20 v. 2016, Revised on 10 Dec. 2016, Accepted on 31 Dec DOI: /wjpps *Corresponding Author Dr. B. Parijatha School of Pharmacy, Nalla Narasimha Reddy Education Society s Group of Institutions, Hyderabad, Telangana, INDIA. ABSTRACT A rapid and sensitive liquid chromatography-mass spectroscopic method was developed for monitoring plasma levels of Amoxicillin and Clavulanate using Amoxicillin D4 as internal standard and validated for applicability for pharmacokinetic studies. The extraction of Amoxicillin and Clavulanate in human plasma involves solid phase extraction. The samples were chromatographed on Kromasil C 18, 100mm, 4.6 mm, 5μm (Make: Akzonobel) column using a mobile phase consisting of HPLC grade Acetonitrile:5mM Ammonium Acetate (80:20, v/v), injection volume was 15μl, needle rinsing volume was 1000μl and flow rate 1.000mL/min. The run time of sample was 2.20 minutes. Retention time for Clavulanate is 0.80 ± 0.5 minutes and for Amoxicillin it is 0.80 ± 0.5 minutes. Detection of analytes was performed on LCMS system in multiple reactions monitoring (MRM) mode by using API 4000 in negative mode. The MS ion transitions monitored were (product ion) for Amoxicillin, (parent ion) (product ion) for Clavulanate, (parent ion) (product ion) for Amoxicillin D4. The linearity was obtained over concentration range of ng/ml to ng/ml for Amoxicillin and ng/ml to ng/ml for Clavulanate. KEYWORDS: Amoxicillin, Clavulanate, LC-MS method, Method development, Method Validation. Vol 6, Issue 01,

2 INTRODUCTION Amoxicillin Amoxicillin is a broad-spectrum semi synthetic antibiotic [1] similar to ampicillin except that its resistance to gastric acid permits higher serum levels with oral administration. Amoxicillin is commonly prescribed with clavulanic acid (a beta lactamase inhibitor) as it is susceptible to beta-lactamase degradation. Figure.1: Molecular Structure of Amoxicillin. Chemical Formula: C 16 H 19 N 3 O 5 S IUPAC Name: 6-{[2-amino-2-(4-hydroxyphenyl)-acetyl] amino}-3, 3-dimethyl-7-oxo-4- thia-1-azabicyclo [3.2.0] heptane-24-carboxylic acid Molecular weight: Physicochemical Properties Solubility: Soluble in Methanol and Water. Categories: Antibiotic MECHANISM OF ACTION Amoxicillin prevents cell wall synthesis by binding to enzymes called penicillin binding proteins and concentration-independent bactericidal activity. Clavulanic acid [2] is a β-lactam structurally related to the penicillin s and possesses the ability to inactivate a wide variety of β-lactamase by blocking the active sites of these enzymes. Combination of Amoxicillin and Clavulanate potassium may prevent Amoxicillin Vol 6, Issue 01,

3 Hydrolyzed by β-lactamase. Clavulanate History Clavulanic acid is in the form its salts and esters. The acid is a suicide inhibitor of bacterial beta-lactamase enzymes from Streptomyces clavuligerus. [3] Administered alone, it has only weak antibacterial activity against most organisms, but given in combination with beta-lactam antibiotics prevents antibiotic inactivation by microbial lactamase. Clavulanic acid is a β-lactamase inhibitor combined with penicillin group antibiotics to overcome certain types of antibiotic resistance. Figure.2: Molecular Structure of Amoxicillin. Chemical Formula C 8 H 9 NO 5 IUPAC Name: 3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-aza-bicyclo [3.2.0] heptane-2- carboxylic acid. Molecular weight : Physicochemical Properties Solubility: Soluble in Methanol and Water Categories: β-lactamase inhibitor Half life : 1.0 hour Mechanism of action Clavulanate is a beta-lactam structurally related to the penicillin s. Clavulanic acid is used in conjunction [4] with Amoxicillin for the treatment of bronchitis and urinary tract, skin, and soft tissue infections caused by beta-lactamase producing organisms. Vol 6, Issue 01,

4 MATERIALS AND METHODS Table 1: List of chemicals. Reagents/Materials Methanol (HPLC grade) Ammonium acetate (AR grade) Acetonitrile (HPLC grade) HPLC grade water Milli-Q water Manufacturer/Supplier JT Baker Merck JT Baker Rankem In house Table 2: List of equipment. S.. Name of equipment Make Model 1 Analytical Balance Sartorius CPA Micro Balance Sartorius SE-Z 3 Ph Meter Drjon 3 STAR 4 Reciprocating Shaker Orbitek SCIGENICS 5 Refrigerate Centrifuge Heraens MEGAFUSE20 R 6 Turbo Evaporator Zymark BE-TE-01 7 Positive processor Pressure Orochen SZYPRESS 48 8 HPLC MS-MS Shimadzu API 4000 Finalized Chromatographic Parameters Column Mobile phase Diluent Rinsing solution Flow rate Split : 50:50 Sample Cooler Temperature : 5 C Column Oven Temperature : N/AP Injection volume : 20 µl Needle Rinsing Volume : 500 µl Rinsing Mode Retention time : Kromasil C18, 100*4.6 mm, 5µm (Make: Akzonobel) : HPLC grade Acetonitrile: 5mM ammonium acetate (80:20, v/v) :HPLC grade Acetonitrile: Milli Q/HPLC grade water (60:40, v/v) : HPLC grade Acetonitrile: Milli Q water (60:40 v/v) : 1.0 ml/minute (with splitter) : Before and after aspiration : Amoxicillin 0.80 ± 0.5 minutes : Clavulanate 0.80 ± 0.5 minutes : Amoxicillin-d ± 0.5 minutes Run Time : 2.20 minutes Vol 6, Issue 01,

5 a) 5mM Ammonium acetate (w/v) buffer About mg of ammonium acetate [6] was transferred to a 1000 ml reagent bottle containing 1000 ml of Milli Q/HPLC grade water. Mixed well and sonicated in an ultrasonicator for 5 minutes. The buffer solution was stored at room temperature (20±5 C) and used within 4 days from the date of preparation. b) Mobile Phase (20:80 v/v) 400 ml of 5mM ammonium acetate was transferred to a 2000 ml reagent bottle and 1600 ml of HPLC grade Acetonitrile was added to it. It was mixed well, sonicated in an ultrasonicator for 5 minutes. The mobile phase was stored at room temperature (20±5 C) and used within 7 days from the date of preparation. c) Diluent (v/v) A mixture of HPLC grade Acetonitrile and Milli Q/HPLC grade water [7] was prepared in the volume ratio of 60:40 as diluent. It was then sonicated in an ultrasonicator for 5 minutes. The diluent was stored at room temperature (20±5 C) and used within 7 days from the date of preparation. d) Rinsing solution (v/v) Diluent was used as rinsing solution. Sample Preparation The samples were thawed at room temperature and vortexed to ensure complete mixing of the contents. 250 µl of the plasma sample was pipetted into 5 ml RIA [8] vial tubes and 25 µl of ng/ml Amoxicillin-d4 dilutions was added to it and vortexed, except in blank plasma samples where 25 µl diluent was added and vortexed. Then 1 ml of Acetonitrile was added and vortexed. The samples were centrifuged at 4000 rpm for 20 minutes at 4 C. Then supernatant layer was transferred to prelabelled auto sampler loading vials and injected. Biological matrix Eight lots of K 2 -EDTA human plasma including one lipemic and one hemolytic plasma were screened for selectivity test. All human plasma lots including hemolytic and lipemic plasma were found free of any significant interference for Amoxicillin and Clavulanate and Internal Standard. All the above screened plasma lots were pooled and used to prepare calibration standards, quality control samples and DIQC samples. Vol 6, Issue 01,

6 Selectivity test was performed before bulk spiking. After bulk spiking, aliquots of 400 µl for CCs and 400 µl for QCs of spiked plasma samples were pipetted out into a prelabelled micro centrifuge tubes and then all the bulk spiked samples were stored in deep freezer at 70 C, except twelve replicates each of LQC and HQC, which were stored in deep freezer at 20 C for generation of stability data at 20 C. Stock Solutions Amoxicillin and Clavulanate Stock Solution Weigh about mg of Amoxicillin working standards separately and transfer to a 5 ml clean volumetric flask and dissolve in Milli Q/HPLC grade water and the volume is made up with the same to produce a solution of mg/ml Similarly, weigh about mg of Clavulanate working standards separately and transfer to a 5 ml clean volumetric flask and dissolve in Milli Q/HPLC grade water and the volume is made up with the same to produce a solution of mg/ml The stock solution was stored in refrigerator at 2 8 C and used for maximum of 6 days. The stock solution of Amoxicillin and Clavulanate were prepared separately. The stock solutions [10] were diluted to suitable concentrations using a mixture of Acetonitrile and Milli Q water (Diluent) in the ratio of (60:40 v/v) for spiking into plasma to obtain calibration curve (CC) standards, quality control (QC) samples and DIQC samples. All other final dilutions (system suitability test, aqueous mixture and recovery samples) were prepared in mobile phase. Amoxicillin D4 Stock Solution (Internal Standard) Weigh about mg of Amoxicillin-d4 working standards separately and transfer to a 2mL clean volumetric flask, dissolve in Milli Q/HPLC grade water and the volume is made up with the same to produce a solution of mg/ml The stock solution was stored in refrigerator at 2 8 C and used for maximum of 6 days. The stock solutions were diluted to suitable concentration using diluent for internal standard dilution. Calibration Curve Standards and Quality Control Samples Calibration curve standard consisting of a set of nine non-zero concentrations ranging from ng/ml to ng/ml of Amoxicillin and ng/ml to ng/ml of Vol 6, Issue 01,

7 Clavulanate were prepared. Prepared quality control samples consisted of concentrations of ng/ml (LLOQ QC), ng/ml (LQC), ng/ml (MQC1), ng/ml (MQC2), and ng/ml (HQC) for Amoxicillin and ng/ml (LLOQ QC), ng/ml (LQC), ng/ml (MQC1), ng/ml (MQC2), and ng/ml (HQC) for Clavulanate. System Suitability Solution A mixture of analytes and internal standard were prepared for system suitability test. The system suitability test solution was injected as an aqueous mixture. Aqueous samples are prepared as per recovery basis. 25 µl of each analyte ( ng/ml of Amoxicillin and ng/ml of Clavulanate) and 50 µl of combined dilution of internal standard ( ng/ml of Amoxicillin-d4) were mixed with 2450 µl of mobile phase to prepare the system suitability sample. Mass spectroscopic conditions MRM mode [9] was used for detection of both analyte and IS as followed in table 1a various mass spectrometric detection dependent parameters for Amoxicillin, Clavulanate and IS were selected as mentioned in table 1b. Table.3a: MRM modes for analytes and IS. Analyte Parent ion (m/z) Product ion (m/z) Amoxicillin Clavulanate Amoxicillin d Table.3b: Mass spectroscopic conditions for analyte and IS. Parameter Amoxicillin Clavulanate Amoxicillin-d4 Ionization mode Negative Negative Negative Detection m/z (parent) and (parent) and (parent) and (product) (product) (product) Ion Spray Voltage (IS) V V V Temperature (TEM 0 C) Curtain Gas (CUR) psi psi psi Collision Gas (CAD) 8.00 psi 8.00 psi 8.00 psi NEB 6.00 psi 6.00 psi 6.00 psi Declustering Potential (DP) V V V Collision Energy (CE) V V V Collision Cell Exit Potential (CXP) V V V Focusing Potential (FP) V V V Entrance Potential (EP) V V V Vol 6, Issue 01,

8 RESULTS AND DISCUSSION Method development and optimization Linearity A regression equation with a weighting factor of 1/ (concentration ratio) 2 of drug to IS concentration was judged to produce the best fit for the concentration-detector response relationship for Amoxicillin and Clavulanate in human plasma. The representative calibration curves for regression analysis are illustrated in Figure 3 & 4 for Amoxicillin and Clavulanate, respectively. Correlation coefficient (r) was greater than 0.99 in the concentration range of ng/ml to ng/ml for Amoxicillin and ng/ml to ng/ml for Clavulanate these results are given in the tables 2a, 2b and 2c. Table.4a: Concentration-response Linearity Data for Amoxicillin. AMCL minal Concentration (ng/ml) STD-A STD-B STD-C STD-D STD-E STD-F STD-G STD-H STD-I CC# Accuracy Accuracy Accuracy Mean S.D C.V minal N Table 4b: Concentration-response Linearity Data for Clavulanate. minal Concentration (ng/ml) AMCL STD-A STD-B STD-C STD-D STD-E STD-F STD-G STD-H STD-I CC# Accuracy Accuracy Accuracy Mean Vol 6, Issue 01,

9 PA BATCH-1 AND SENSITIVITY.rdb (Amoxicillin): "Linear" Regression ("1 / (x * x)" weighting): y = x (r = ) e4 1.1e4 1.2e4 1.3e4 1.4e4 1.5e4 1.6e4 1.7e4 1.8e4 Analyte Conc. / IS Conc. PA BATCH-1 AND SENSITIVITY.rdb (Clavulanate): "Linear" Regression ("1 / (x * x)" weighting): y = x (r = ) Analyte Conc. / IS Conc. Parijatha et al. S.D C.V minal N Table.4c: Linearity values for Amoxicillin and Clavulanate. AMCL CC# Slope Intercept R r Analyte Area / IS Area Figure 3: A Representative Calibration Curve for Regression Analysis of Amoxicillin Analyte Area / IS Area Figure 4: A Representative Calibration Curve for Regression Analysis of Clavulanate Vol 6, Issue 01,

10 Sensitivity The lowest limit of reliable quantification for Amoxicillin and Clavulanate in human plasma was set at the concentration of the LLOQ, ng/ml and ng/ml, respectively. The precision and accuracy for Amoxicillin at this concentration was found to be Similarly, the precision and accuracy for Clavulanate at this concentration was found to be 3.46 and These results are given in the table 3a and 3b. Table 5a: Within Batch Precision and Accuracy for Sensitivity of Amoxicillin. -AMCL minal concentration (ng/ml) SEN-LLOQ Accuracy Mean S.D C.V 2.83 minal N 6 Table 5b: Within Batch Precision and Accuracy for Sensitivity of Clavulanate. Auto sampler Stability AMCL minal Concentration (ng/ml) SEN- LLOQ Accuracy Mean S.D C.V 3.46 minal N 6 In assessing the auto sampler stability of Amoxicillin and Clavulanate, six sets of QC samples (LQC and HQC) were processed and placed in the auto sampler. [12] These samples were injected after a period of 54 hours. Refer, Table 4a, 4b. Results demonstrate that the Vol 6, Issue 01,

11 processed samples were stable for 54 hours. The percent nominal of Amoxicillin ranged from to and the precision ranged from 1.32 to The percent nominal of Clavulanate ranged from to and the precision ranged from 2.50 to Table 6a: Auto sampler Stability Data of Amoxicillin for 53 hours. AMCL minal Concentration (ng/ml) LQC HQC QC# Accuracy Accuracy Mean S.D C.V. () minal N 6 6 Table 6b: Auto sampler Stability Data of Clavulanate for 53 hours. Data processing AMCL minal Concentration (ng/ml) LQC HQC QC# Accuracy Accuracy Mean S.D C.V.() minal N 6 6 The chromatograms were acquired and processed by peak area ratio method using the Analyst software. The concentration of the unknown was calculated from the following equation using regression analysis of spiked standard with the reciprocal of the square of ratio of the drug concentration to internal standard concentration as a weighting factor [1/ (concentration ratio) 2 ]. Vol 6, Issue 01,

12 Sample Name: "Blank" Sample ID: "" File: "002.wiff" Peak Name: "Amoxicillin" Mass(es): "363.9/223.1 amu" Sample Type: Double Blank Concentration: ng/ml Acq. Time: 15:21:50 ise Threshold: cps Area Threshold: cps Sep Expected RT: min Use Relative RT: Retention Time: 0.79 min Area: 1223 counts 1.76e+002 cps Start Time: min End Time: min Sample Name: "Blank" Sample ID: "" File: "002.wiff" Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu" Sample Type: Double Blank Concentration: 1.00 ng/ml Acq. Time: 15:21:50 Sample Name: "AQM(MV-279-SST-03)" Sample ID: "" File: "001.wiff" Peak Name: "Amoxicillin" Mass(es): "363.9/223.1 amu" Sample Type: Unknown Concentration: N/A Calculated Conc: ng/ml 1.7e5 1.6e5 Acq. Time: 15:18:55 1.6e5 1.5e5 ise Threshold: cps 1.5e5 Area Threshold: cps 1.4e5 Sep e5 1.3e5 1.3e5 Expected RT: min 1.2e5 Use Relative RT: 1.2e5 Retention Time: 0.80 min 1.1e5 Area: counts 1.67e+005 cps 1.1e5 Start Time: min End Time: 1.01 min 1.0e5 9.5e4 9.0e4 8.5e4 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Sample Name: "AQM(MV-279-SST-03)" Sample ID: "" File: "001.wiff" Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu" Sample Type: Unknown Concentration: 1.00 ng/ml 9.0e4 Acq. Time: 15:18:55 ise Threshold: cps Area Threshold: cps Sep Expected RT: min Use Relative RT: Retention Time: 0.79 min Area: counts 9.36e+004 cps Start Time: min End Time: min e4 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Parijatha et al. y = mx + c Where, y = peak area ratio of Amoxicillin/Clavulanate to respective internal standard m = slope of the calibration curve x = concentration ratio of Amoxicillin/Clavulanate to respective internal standard ng/ml c = y-axis intercept of the calibration. Figure 5: Chromatogram of an Aqueous Standard and Internal Standard Mixture of Amoxicillin Figure 6: Chromatogram of Blank Plasma Sample of Amoxicillin. Vol 6, Issue 01,

13 Sample Name: "Blank+IS" Sample ID: "" File: "003.wiff" Peak Name: "Amoxicillin" Mass(es): "363.9/223.1 amu" Sample Type: Blank Concentration: ng/ml Acq. Time: 15:24:45 ise Threshold: cps Area Threshold: cps Sep Expected RT: min Use Relative RT: Retention Time: 0.78 min Area: 1622 counts 2.62e+002 cps Start Time: min End Time: min Sample Name: "Blank+IS" Sample ID: "" File: "003.wiff" Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu" Sample Type: Blank Concentration: 1.00 ng/ml Acq. Time: 15:24:45 ise Threshold: cps Area Threshold: cps Sep Expected RT: min Use Relative RT: Retention Time: 0.79 min Area: counts 1.17e+005 cps Start Time: min End Time: min e5 1.10e5 1.05e5 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e Sample Name: "AQM(MV-279-SST-03)" Sample ID: "" File: "001.wiff" Peak Name: "Clavulanate" Mass(es): "198.0/135.8 amu" Sample Type: Unknown Concentration: N/A Calculated Conc: Acq. Time: 15:18:55 ng/ml ise Threshold: cps Area Threshold: cps Sep Expected RT: min Use Relative RT: Retention Time: 0.80 min Area: counts 1.04e+005 cps Start Time: min End Time: min 1.00e5 9.50e4 9.00e4 8.50e4 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 2.00e4 1.50e4 1.00e Sample Name: "AQM(MV-279-SST-03)" Sample ID: "" File: "001.wiff" Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu" Sample Type: Unknown Concentration: 1.00 ng/ml Acq. Time: 15:18:55 ise Threshold: cps Area Threshold: cps Sep Expected RT: min Use Relative RT: Retention Time: 0.79 min Area: counts 9.36e+004 cps Start Time: min End Time: min 9.0e4 8.5e4 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e Sample Name: "Blank" Sample ID: "" File: "002.wiff" Peak Name: "Clavulanate" Mass(es): "198.0/135.8 amu" Sample Type: Double Blank Concentration: ng/ml Acq. Time: 15:21:50 ise Threshold: cps Area Threshold: cps Sep Expected RT: min Use Relative RT: Retention Time: 0.73 min Area: 402 counts 8.60e+001 cps Start Time: min End Time: min Sample Name: "Blank" Sample ID: "" File: "002.wiff" Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu" Sample Type: Double Blank Concentration: 1.00 ng/ml Acq. Time: 15:21: Parijatha et al. Figure 7: Chromatogram of Blank Plasma with Internal Standard Sample of Amoxicillin. Figure 8: Chromatogram of an Aqueous Standard and Internal Standard Mixture of Clavulanate. Figure 9: Chromatogram of Blank Plasma Sample of Clavulanate Vol 6, Issue 01,

14 Limits of detection and quantification The limit of detection (LOD) is defined as the lowest concentration of an analyte that can be Readily detected but not necessarily quantified. It is usually regarded as the amount for which the signal-to-noise ratio (SNR) is 3:1. The limit of quantization (LOQ) is defined as the lowest Concentration of an analyte that can A Representative Chromatogram of an Aqueous Standard and Internal Standard Mixture of Clavulanate. CONCLUSION A high performance liquid chromatography mass spectrometric method for the estimation of Amoxicillin and Clavulanate in human plasma in negative ion mode was developed and validated using Amoxicillin D4 as internal standard (IS). Sample preparation was accomplished by Solid phase extraction technique. The reconstituted samples were chromatographed on Kromasil C18, 100*4.6 mm, 5µm (Make: Akzonobel) column using a mobile phase consisting of HPLC grade Acetonitrile: 5mM ammonium acetate (80:20, v/v). The method was validated over a concentration range of ng/ml to ng/ml for Amoxicillin and ng/ml to ng/ml for Clavulanate. This validation report provides the results of selectivity, and sensitivity determinations, calibration standards and quality control samples data, precision and accuracy data, the results of recovery, various stabilities and dilution along with all pertinent supporting documentation. REFERENCES 1. Avinash Ghaikwad, Sumith Gavali et.al. An LC MS MS method for the simultaneous quantification of amoxicillin and clavulanic acid in human plasma and its pharmacokinetic applications. Journal of Pharmacy Research. 2013; 6(8): Durga Mallikarjuna Roan Tippa* and Narendra Singh, Development and Validation of Stability Indicating HPLC Method for Simultaneous Estimation of Amoxicillin and Clavulanic Acid in Injection American Journal of Analytical Chemistry., 2010; 1: Chaitanya Krishna and Chelladurai, a novel and high-throughput method for the simultaneous determination of amoxicillin and Clavulanic acid in human plasma by liquid chromatography coupled with tandem mass spectrometry. International Journal of Pharmacy and Pharmaceutical Sciences, 2012; Vol 6, Issue 01,

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