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

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES Parijatha et al. SJIF Impact Factor 6.041 Volume 6, Issue 1, 1540-1554 Research Article ISSN 2278 4357 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. 2016 DOI: 10.20959/wjpps20171-8445 *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 100-5 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 363.9 223.10 (product ion) for Amoxicillin, 198.00 (parent ion) 135.80 (product ion) for Clavulanate, 368.00(parent ion) 227.10(product ion) for Amoxicillin D4. The linearity was obtained over concentration range of 153.596 ng/ml to 18034.104 ng/ml for Amoxicillin and 48.800 ng/ml to 5729.720 ng/ml for Clavulanate. KEYWORDS: Amoxicillin, Clavulanate, LC-MS method, Method development, Method Validation. www.wjpps.com Vol 6, Issue 01, 2017. 1540

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: 365.4 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 www.wjpps.com Vol 6, Issue 01, 2017. 1541

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 : 199.16 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. www.wjpps.com Vol 6, Issue 01, 2017. 1542

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 CPA2250 2 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 100-5 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-d4 0.80 ± 0.5 minutes Run Time : 2.20 minutes www.wjpps.com Vol 6, Issue 01, 2017. 1543

a) 5mM Ammonium acetate (w/v) buffer About 385.4 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 49848.608 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. www.wjpps.com Vol 6, Issue 01, 2017. 1544

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 5.000 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 1.000 mg/ml Similarly, weigh about 5.000 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 1.000 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 2.000 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 1.000 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 153.596 ng/ml to 18034.104 ng/ml of Amoxicillin and 48.800 ng/ml to 5729.720 ng/ml of www.wjpps.com Vol 6, Issue 01, 2017. 1545

Clavulanate were prepared. Prepared quality control samples consisted of concentrations of 154.417 ng/ml (LLOQ QC), 456.854 ng/ml (LQC), 1903.558 ng/ml (MQC1), 9064.563 ng/ml (MQC2), and 14620.263 ng/ml (HQC) for Amoxicillin and 48.845 ng/ml (LLOQ QC), 144.512 ng/ml (LQC), 602.132 ng/ml (MQC1), 2867.295 ng/ml (MQC2), and 4624.670 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 (176853.195 ng/ml of Amoxicillin and 56203.312 ng/ml of Clavulanate) and 50 µl of combined dilution of internal standard (49848.608 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 363.90 223.10 Clavulanate 198.00 198.00 Amoxicillin d4 368.00 227.10 Table.3b: Mass spectroscopic conditions for analyte and IS. Parameter Amoxicillin Clavulanate Amoxicillin-d4 Ionization mode Negative Negative Negative Detection m/z 363.90 (parent) and 198.00 (parent) and 368.00 (parent) and 223.10 (product) 135.80 (product) 227.10 (product) Ion Spray Voltage (IS) -4600.00 V -4600.00 V -4600.00 V Temperature (TEM 0 C) 550.00 550.00 550.00 Curtain Gas (CUR) 12.00 psi 12.00 psi 12.00 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) -35.00 V -13.00 V -13.00 V Collision Energy (CE) -14.00 V -11.00 V -14.00 V Collision Cell Exit Potential (CXP) -4.00 V -9.00 V -4.00 V Focusing Potential (FP) -130.00 V -60.00 V -75.00 V Entrance Potential (EP) -10.00 V -10.00 V -10.00 V www.wjpps.com Vol 6, Issue 01, 2017. 1546

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 153.596 ng/ml to 18034.104 ng/ml for Amoxicillin and 48.800 ng/ml to 5729.720 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# 153.596 307.193 903.509 1807.017 3614.034 7228.069 10820.462 14427.283 18034.104 1 153.015 307.790 918.836 1804.766 3577.067 7523.901 10939.812 13838.119 17767.693 Accuracy 99.62 100.19 101.70 99.88 98.98 104.09 101.10 95.92 98.52 2 154.443 302.997 900.122 1832.155 3689.655 7318.908 10801.108 13913.049 18068.742 Accuracy 100.55 98.63 99.63 101.39 102.09 101.26 99.82 96.44 100.19 3 150.894 316.030 917.539 1843.927 3561.625 7110.350 10741.782 14266.660 18071.322 Accuracy 98.24 102.88 101.55 102.04 98.55 98.37 99.27 98.89 100.21 Mean 152.7840 308.9390 912.1657 1826.9493 3609.4490 7317.7197 10827.5673 14005.9427 17969.2523 S.D. 1.45805 5.38237 8.53260 16.40569 57.06351 168.83358 82.98210 186.87567 142.52786 C.V. 0.95 1.74 0.94 0.90 1.58 2.31 0.77 1.33 0.79 minal 99.47 100.57 100.96 101.10 99.87 101.24 100.07 97.08 99.64 N 3 3 3 3 3 3 3 3 3 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# 48.800 97.600 287.059 574.118 1148.236 2296.472 3437.832 4583.776 5729.720 1 47.746 101.778 288.388 578.472 1110.925 2332.502 3446.773 4433.234 5807.736 97.84 104.28 100.46 100.76 96.75 101.57 100.26 96.72 101.36 Accuracy 2 49.177 97.102 277.994 575.015 1135.966 2320.732 3527.345 4382.886 5989.284 100.77 99.49 96.84 100.16 98.93 101.06 102.60 95.62 104.53 Accuracy 3 48.294 100.391 284.554 569.044 1098.001 2263.025 3433.551 4576.402 6076.441 98.96 102.86 99.13 99.12 95.63 98.54 99.88 99.84 106.05 Accuracy Mean 48.4057 99.7570 283.6453 574.1770 1114.9640 2305.4197 3469.2230 4464.1740 5957.8203 www.wjpps.com Vol 6, Issue 01, 2017. 1547

PA BATCH-1 AND SENSITIVITY.rdb (Amoxicillin): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000199 x + 0.00204 (r = 0.9997) 3.6 3.5 3.4 3.3 3.2 3.1 3.0 2.9 2.8 2.7 2.6 2.5 2.4 2.3 2.2 2.1 2.0 1.9 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 1000.0 2000.0 3000.0 4000.0 5000.0 6000.0 7000.0 8000.0 9000.0 1.0e4 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 = 0.000413 x + 0.000427 (r = 0.9996) 2.4 2.4 2.4 2.3 2.3 2.2 2.1 2.1 2.1 2.0 2.0 1.9 1.9 1.8 1.8 1.7 1.7 1.6 1.6 1.5 1.5 1.4 1.4 1.3 1.3 1.2 1.2 1.1 1.1 1.0 1.0 0.9 0.9 0.8 0.8 0.7 0.7 0.6 0.6 0.5 0.5 0.4 0.4 0.3 0.3 0.2 0.2 0.1 0.1 0.0 500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500 Analyte Conc. / IS Conc. Parijatha et al. S.D. 0.58952 1.96090 4.29170 3.89431 15.76009 30.36022 41.45142 81.97590 111.93172 C.V. 1.22 1.97 1.51 0.68 1.41 1.32 1.19 1.84 1.88 minal 99.19 102.21 98.81 100.01 97.10 100.39 100.91 97.39 103.98 N 3 3 3 3 3 3 3 3 3 Table.4c: Linearity values for Amoxicillin and Clavulanate. AMCL CC# Slope Intercept R r 2 1 0.0004 0.0004 0.9996 0.9992 2 0.0004-0.0003 0.9995 0.9990 3 0.0004 0.0004 0.9995 0.9990 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 www.wjpps.com Vol 6, Issue 01, 2017. 1548

Sensitivity The lowest limit of reliable quantification for Amoxicillin and Clavulanate in human plasma was set at the concentration of the LLOQ, 153.596 ng/ml and 48.800 ng/ml, respectively. The precision and accuracy for Amoxicillin at this concentration was found to be 2.83 88.41. Similarly, the precision and accuracy for Clavulanate at this concentration was found to be 3.46 and 91.77. 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 153.596 Accuracy 1 132.020 85.95 2 142.591 92.84 3 137.610 89.59 4 135.036 87.92 5 134.464 87.54 6 133.616 86.60 Mean 135.7898 S.D 3.84182 C.V 2.83 minal 88.41 N 6 Table 5b: Within Batch Precision and Accuracy for Sensitivity of Clavulanate. Auto sampler Stability AMCL minal Concentration (ng/ml) SEN- LLOQ 48.800 Accuracy 1 43.773 89.70 2 46.979 96.27 3 44.578 91.35 4 45.654 93.55 5 42.509 87.11 6 45.218 92.66 Mean 44.7852 S.D 1.54942 C.V 3.46 minal 91.77 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 www.wjpps.com Vol 6, Issue 01, 2017. 1549

processed samples were stable for 54 hours. The percent nominal of Amoxicillin ranged from 94.32 to 95.05 and the precision ranged from 1.32 to 1.81. The percent nominal of Clavulanate ranged from 104.00 to106.47 and the precision ranged from 2.50 to 4.22. Table 6a: Auto sampler Stability Data of Amoxicillin for 53 hours. AMCL minal Concentration (ng/ml) LQC HQC QC# 456.854 Accuracy 14620.263 Accuracy 95 447.113 97.87 13938.522 95.34 96 429.983 94.12 13552.794 92.70 97 437.624 95.79 13635.386 93.26 98 425.598 93.16 13725.738 93.88 99 436.636 95.57 14024.834 95.93 100 428.546 93.80 13857.705 94.78 Mean 434.2500 13789.1632 S.D. 7.85027 182.25660 C.V. () 1.81 1.32 minal 95.05 94.32 N 6 6 Table 6b: Auto sampler Stability Data of Clavulanate for 53 hours. Data processing AMCL minal Concentration (ng/ml) LQC HQC QC# 144.512 Accuracy 4624.670 Accuracy 95 153.847 106.46 4773.819 103.23 96 152.581 105.58 4834.706 104.54 97 149.652 103.56 4891.427 105.77 98 137.840 95.38 5110.595 110.51 99 152.990 105.87 5020.525 108.56 100 154.826 107.14 4912.834 106.23 Mean 150.2893 4923.9843 S.D. 6.34312 123.16509 C.V.() 4.22 2.50 minal 104.00 106.47 N 6 6 The chromatograms were acquired and processed by peak area ratio method using the Analyst 1.4.2 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 ]. www.wjpps.com Vol 6, Issue 01, 2017. 1550

Sample Name: "Blank" Sample ID: "" File: "002.wiff" Peak Name: "Amoxicillin" Mass(es): "363.9/223.1 amu" Sample Type: Double Blank Concentration: 0.000 ng/ml Acq. Time: 15:21:50 ise Threshold: 20.00 cps Area Threshold: 200.00 cps Sep. 1.00 Expected RT: 0.752 min Use Relative RT: Retention Time: 0.79 min Area: 1223 counts 1.76e+002 cps Start Time: 0.666 min End Time: 0.861 min 220 210 200 190 180 170 160 150 140 130 120 110 100 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: 9080.721 ng/ml 1.7e5 1.6e5 Acq. Time: 15:18:55 1.6e5 1.5e5 ise Threshold: 20.00 cps 1.5e5 Area Threshold: 200.00 cps 1.4e5 Sep. 1.00 1.4e5 1.3e5 1.3e5 Expected RT: 0.752 min 1.2e5 Use Relative RT: 1.2e5 Retention Time: 0.80 min 1.1e5 Area: 991100 counts 1.67e+005 cps 1.1e5 Start Time: 0.646 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 1.0e4 5000.0 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: 25.00 cps Area Threshold: 200.00 cps Sep. 1.00 Expected RT: 0.746 min Use Relative RT: Retention Time: 0.79 min Area: 547651 counts 9.36e+004 cps Start Time: 0.636 min End Time: 0.974 min 0.0 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 1.0e4 5000.0 0.0 90 80 70 60 50 40 30 20 10 0 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 0.78 0.88 0.80 0.79 2.05 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. www.wjpps.com Vol 6, Issue 01, 2017. 1551

Sample Name: "Blank+IS" Sample ID: "" File: "003.wiff" Peak Name: "Amoxicillin" Mass(es): "363.9/223.1 amu" Sample Type: Blank Concentration: 0.000 ng/ml Acq. Time: 15:24:45 ise Threshold: 20.00 cps Area Threshold: 200.00 cps Sep. 1.00 Expected RT: 0.752 min Use Relative RT: Retention Time: 0.78 min Area: 1622 counts 2.62e+002 cps Start Time: 0.666 min End Time: 0.861 min 320 310 300 290 280 270 260 250 240 230 220 210 200 190 180 170 160 150 140 130 120 110 100 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: 25.00 cps Area Threshold: 200.00 cps Sep. 1.00 Expected RT: 0.746 min Use Relative RT: Retention Time: 0.79 min Area: 673657 counts 1.17e+005 cps Start Time: 0.636 min End Time: 0.974 min 90 80 70 60 50 40 30 20 10 0 1.15e5 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 1.00e4 5000.00 0.00 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: 2928.909 Acq. Time: 15:18:55 ng/ml ise Threshold: 10.00 cps Area Threshold: 100.00 cps Sep. 1.00 Expected RT: 0.741 min Use Relative RT: Retention Time: 0.80 min Area: 662517 counts 1.04e+005 cps Start Time: 0.636 min End Time: 0.994 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 1.00e4 5000.00 0.00 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: 25.00 cps Area Threshold: 200.00 cps Sep. 1.00 Expected RT: 0.746 min Use Relative RT: Retention Time: 0.79 min Area: 547651 counts 9.36e+004 cps Start Time: 0.636 min End Time: 0.974 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 1.0e4 5000.0 Sample Name: "Blank" Sample ID: "" File: "002.wiff" Peak Name: "Clavulanate" Mass(es): "198.0/135.8 amu" Sample Type: Double Blank Concentration: 0.000 ng/ml Acq. Time: 15:21:50 ise Threshold: 10.00 cps Area Threshold: 100.00 cps Sep. 1.00 Expected RT: 0.741 min Use Relative RT: Retention Time: 0.73 min Area: 402 counts 8.60e+001 cps Start Time: 0.656 min End Time: 0.830 min 135 130 125 120 115 110 105 100 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 0.0 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 130 125 120 115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 0.38 0.78 0.89 0.97 1.05 0.79 0.80 0.79 1.17 1.30 1.41 1.47 1.61 1.75 1.97 2.17 0.78 0.88 2.05 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 www.wjpps.com Vol 6, Issue 01, 2017. 1552

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 100-5 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 153.596 ng/ml to 18034.104 ng/ml for Amoxicillin and 48.800 ng/ml to 5729.720 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): 804 812. 2. 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: 95-101. 3. 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; 0975-1491. www.wjpps.com Vol 6, Issue 01, 2017. 1553

4. Ajitha, A Thenmozhi*, D Sridhar an, V Rajamanickam1, Rapid and sensitive LCMS/MS method for the simultaneous estimation of Amoxicillin and Clavulanic acid in human plasma. Asian Journal of Pharmaceutical and Clinical Research, 2010; 0974 41. 5. Akhilesh Gupta, Rajkumar1, Swati Rawat2, Mayuri Gandhi3 and Rahul, Named. Simultaneous estimation of amoxicillin and potassium Clavulanate in injection formulation by simultaneous equation method and its validation Journal of Pharmacy Research., 2011; 4(4): 1244-1245. 6. Hartmann c, Smeyers-Verbeke, Massart DL, MC Dowel RD. Validation of Bioanalytical chromatographic methods. Journal of Pharmaceutical and Biomedical Analysis, 1998; 17(2): 193 218. 7. Roger Cousin, Validation of chromatographic methods in biomedical analysis view point and discussion, Journal of Chromatography 1997; 175-180. 8. Bressolle, F, Bromet-pitit, M. & Audran, M, Validation of Liquid Chromatography and Gas Chromatographic Methods Application to Pharmacokinetics, Journal of Chromatography 1996; 3-10. 9. Bioanalysis by LC MS/MS, J. Pharm. Biomed. Anal, 2007; 44(2): 342-355. 10. Howard Hill., High throughput Bio analytical Sample Preparation Methods and Automation Strategies", 1st Ed, 2009; 1(1): 116-209. 11. Jurgen J.G, John Willey & Sons, Chichester, Mass Spectrometry Instrumentation Interpretation and application, 1st Ed, 2009; 216-389. 12. Stooge DA, Holler FJ, Crouch, Principles of Instrumental Analysis, 5th Ed, 1998; 550-55. www.wjpps.com Vol 6, Issue 01, 2017. 1554