DEVELOPMENT AND VALIDATION OF AMOXICILLIN AND … · History Clavulanic acid is in the form its salts and esters. The acid is a suicide inhibitor of bacterial beta-lactamase enzymes

www.wjpps.com Vol 6, Issue 01, 2017.

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Parijatha et al. World Journal of Pharmacy and Pharmaceutical Sciences

DEVELOPMENT AND VALIDATION OF AMOXICILLIN AND

CLAVULANATE BY USING LC-MS METHOD

B. Parijatha1*

, K. Anitha2, K. Prashanthi

1, D. Santhoshi Priya

1, K. Durga Prasad

2 and

Krishnamohan Chinnala1

1School of Pharmacy, Nalla Narasimha Reddy Education Society’s Group of Institutions,

Hyderabad, Telangana, INDIA.

2Mother Theresa College of Pharmacy, NFC Nagar, 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 C18,

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.

WORLD JOURNAL OF PHARMACY AND PHARMACEUTICAL SCIENCES

SJIF Impact Factor 6.041

Volume 6, Issue 1, 1540-1554 Research Article ISSN 2278 – 4357

Article Received on

20 Nov. 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.


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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: C16H19N3O5S

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


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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 C8H

9NO

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.


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MATERIALS AND METHODS

Table 1: List of chemicals.

Reagents/Materials Manufacturer/Supplier

Methanol (HPLC grade) JT Baker

Ammonium acetate (AR grade) Merck

Acetonitrile (HPLC grade) JT Baker

HPLC grade water Rankem

Milli-Q water In house

Table 2: List of equipment.

S.No. 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 : Kromasil 100-5 C18, 100*4.6 mm, 5µm (Make: Akzonobel)

Mobile phase : HPLC grade Acetonitrile: 5mM ammonium acetate (80:20, v/v)

Diluent :HPLC grade Acetonitrile: Milli Q/HPLC grade water (60:40, v/v)

Rinsing solution : HPLC grade Acetonitrile: Milli Q water (60:40 v/v)

Flow rate : 1.0 mL/minute (with splitter)

Split : 50:50

Sample Cooler Temperature : 5°C

Column Oven Temperature : N/AP

Injection volume : 20 µl

Needle Rinsing Volume : 500 µl

Rinsing Mode : Before and after aspiration

Retention time : Amoxicillin 0.80 ± 0.5 minutes

: Clavulanate 0.80 ± 0.5 minutes

: Amoxicillin-d4 0.80 ± 0.5 minutes

Run Time : 2.20 minutes


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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 K2 -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.


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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


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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

223.10 (product)

198.00 (parent) and

135.80 (product)

368.00 (parent) and

227.10 (product)

Ion Spray Voltage (IS) -4600.00 V -4600.00 V -4600.00 V

Temperature (TEM0C) 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


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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 Nominal 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

%

Nominal 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.


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

%

Accuracy 97.84 104.28 100.46 100.76 96.75 101.57 100.26 96.72 101.36

2 49.177 97.102 277.994 575.015 1135.966 2320.732 3527.345 4382.886 5989.284

%

Accuracy 100.77 99.49 96.84 100.16 98.93 101.06 102.60 95.62 104.53

3 48.294 100.391 284.554 569.044 1098.001 2263.025 3433.551 4576.402 6076.441

%

Accuracy 98.96 102.86 99.13 99.12 95.63 98.54 99.88 99.84 106.05

Mean 48.4057 99.7570 283.6453 574.1770 1114.9640 2305.4197 3469.2230 4464.1740 5957.8203


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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

%

Nominal 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 Slope Intercept R r

2

CC#

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

PA BATCH-1 AND SENSITIVITY.rdb (Amoxicillin): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000199 x + 0.00204 (r = 0.9997)

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 1.8e4Analyte Conc. / IS Conc.

0.0

0.1

0.2

0.3

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2.7

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2.9

3.0

3.1

3.2

3.3

3.4

3.5

3.6

Analyte

Area / IS

Area

Figure 3: A Representative Calibration Curve for Regression Analysis of Amoxicillin

PA BATCH-1 AND SENSITIVITY.rdb (Clavulanate): "Linear" Regression ("1 / (x * x)" weighting): y = 0.000413 x + 0.000427 (r = 0.9996)

500 1000 1500 2000 2500 3000 3500 4000 4500 5000 5500Analyte Conc. / IS Conc.

0.0

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1.4

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2.2

2.3

2.3

2.4

2.4

2.4

Analyte

Area / IS

Area

Figure 4: A Representative Calibration Curve for Regression Analysis of Clavulanate


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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 Nominal 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

%Nominal 88.41

N 6

Table 5b: Within Batch Precision and Accuracy for Sensitivity of Clavulanate.


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

%Nominal 91.77

N 6

Auto sampler Stability

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


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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.


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

% Nominal 95.05 94.32

N 6 6

Table 6b: Auto sampler Stability Data of Clavulanate for 53 hours.


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

% Nominal 104.00 106.47

N 6 6

Data processing

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

].


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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.

Sample Name: "Blank" Sample ID: "" File: "002.wiff"Peak Name: "Amoxicillin" Mass(es): "363.9/223.1 amu"Comment: "" Annotation: ""

Sample Index: 1

Sample Type: Double Blank

Concentration: 0.000 ng/mL

Calculated Conc: N/A

Acq. Date: 16/12/13

Acq. Time: 15:21:50

Modified: No

Proc. Algorithm: Analyst Classic

Bunching Factor: 1

Noise Threshold: 20.00 cps

Area Threshold: 200.00 cps

Num. Smooths: 7

Sep. Width: 0.20

Sep. Height: 1.00

Exp. Peak Ratio: 5.00

Exp. Adj. Ratio: 4.00

Exp. Val. Ratio: 3.00

RT Window: 30.0 sec

Expected RT: 0.752 min

Use Relative RT: No

Int. Type: Base To Base

Retention Time: 0.79 min

Area: 1223 counts

Height: 1.76e+002 cps

Start Time: 0.666 min

End Time: 0.861 min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0

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Sample Name: "Blank" Sample ID: "" File: "002.wiff"Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu"Comment: "" Annotation: ""

Sample Index: 1




Acq. Date: 16/12/13

Acq. Time: 15:21:50

Modified: No

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0

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sity,

cps

0.78

0.88

2.05

Figure 5: Chromatogram of an Aqueous Standard and Internal Standard Mixture of

Amoxicillin

Sample Name: "AQM(MV-279-SST-03)" Sample ID: "" File: "001.wiff"Peak Name: "Amoxicillin" Mass(es): "363.9/223.1 amu"Comment: "" Annotation: ""

Sample Index: 1

Sample Type: Unknown

Concentration: N/A

Calculated Conc: 9080.721 ng/mL

Acq. Date: 16/12/13

Acq. Time: 15:18:55

Modified: No


Bunching Factor: 1



Num. Smooths: 7

Sep. Width: 0.20

Sep. Height: 1.00




RT Window: 30.0 sec


Use Relative RT: No



Area: 991100 counts



End Time: 1.01 min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

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Inte

nsity

, cps

0.80

Sample Name: "AQM(MV-279-SST-03)" Sample ID: "" File: "001.wiff"Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu"Comment: "" Annotation: ""

Sample Index: 1




Acq. Date: 16/12/13

Acq. Time: 15:18:55

Modified: No


Bunching Factor: 1



Num. Smooths: 7

Sep. Width: 0.20

Sep. Height: 1.00




RT Window: 30.0 sec


Use Relative RT: No



Area: 547651 counts



End Time: 0.974 min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

Inte

nsity

, cps

0.79

Figure 6: Chromatogram of Blank Plasma Sample of Amoxicillin.


1552


Sample Name: "Blank+IS" Sample ID: "" File: "003.wiff"Peak Name: "Amoxicillin" Mass(es): "363.9/223.1 amu"Comment: "" Annotation: ""

Sample Index: 1

Sample Type: Blank



Acq. Date: 16/12/13

Acq. Time: 15:24:45

Modified: No


Bunching Factor: 1



Num. Smooths: 7

Sep. Width: 0.20

Sep. Height: 1.00




RT Window: 30.0 sec


Use Relative RT: No



Area: 1622 counts



End Time: 0.861 min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

160

170

180

190

200

210

220

230

240

250

260

270

280

290

300

310

320

Inten

sity,

cps

0.78

0.89 0.97

1.05

1.17

1.301.61

1.471.41

1.750.38

1.97 2.17

Sample Name: "Blank+IS" Sample ID: "" File: "003.wiff"Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu"Comment: "" Annotation: ""

Sample Index: 1

Sample Type: Blank



Acq. Date: 16/12/13

Acq. Time: 15:24:45

Modified: No


Bunching Factor: 1



Num. Smooths: 7

Sep. Width: 0.20

Sep. Height: 1.00




RT Window: 30.0 sec


Use Relative RT: No



Area: 673657 counts



End Time: 0.974 min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

1.05e5

1.10e5

1.15e5

Inten

sity,

cps

0.79

Figure 7: Chromatogram of Blank Plasma with Internal Standard Sample of

Amoxicillin.

Sample Name: "AQM(MV-279-SST-03)" Sample ID: "" File: "001.wiff"Peak Name: "Clavulanate" Mass(es): "198.0/135.8 amu"Comment: "" Annotation: ""

Sample Index: 1


Concentration: N/A

Calculated Conc: 2928.909 ng/mL

Acq. Date: 16/12/13

Acq. Time: 15:18:55

Modified: No


Bunching Factor: 1



Num. Smooths: 7

Sep. Width: 0.20

Sep. Height: 1.00




RT Window: 30.0 sec


Use Relative RT: No



Area: 662517 counts



End Time: 0.994 min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0.00

5000.00

1.00e4

1.50e4

2.00e4

2.50e4

3.00e4

3.50e4

4.00e4

4.50e4

5.00e4

5.50e4

6.00e4

6.50e4

7.00e4

7.50e4

8.00e4

8.50e4

9.00e4

9.50e4

1.00e5

Inten

sity,

cps

0.80

Sample Name: "AQM(MV-279-SST-03)" Sample ID: "" File: "001.wiff"Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu"Comment: "" Annotation: ""

Sample Index: 1




Acq. Date: 16/12/13

Acq. Time: 15:18:55

Modified: No


Bunching Factor: 1



Num. Smooths: 7

Sep. Width: 0.20

Sep. Height: 1.00




RT Window: 30.0 sec


Use Relative RT: No



Area: 547651 counts



End Time: 0.974 min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0.0

5000.0

1.0e4

1.5e4

2.0e4

2.5e4

3.0e4

3.5e4

4.0e4

4.5e4

5.0e4

5.5e4

6.0e4

6.5e4

7.0e4

7.5e4

8.0e4

8.5e4

9.0e4

Inten

sity,

cps

0.79

Figure 8: Chromatogram of an Aqueous Standard and Internal Standard Mixture of

Clavulanate.

Sample Name: "Blank" Sample ID: "" File: "002.wiff"Peak Name: "Clavulanate" Mass(es): "198.0/135.8 amu"Comment: "" Annotation: ""

Sample Index: 1




Acq. Date: 16/12/13

Acq. Time: 15:21:50

Modified: No


Bunching Factor: 1



Num. Smooths: 7

Sep. Width: 0.20

Sep. Height: 1.00




RT Window: 30.0 sec


Use Relative RT: No



Area: 402 counts



End Time: 0.830 min

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

125

130

135

Inte

nsity

, cps

Sample Name: "Blank" Sample ID: "" File: "002.wiff"Peak Name: "Amoxicillin-D4(IS)" Mass(es): "368.0/227.1 amu"Comment: "" Annotation: ""

Sample Index: 1




Acq. Date: 16/12/13

Acq. Time: 15:21:50

Modified: No

0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0Time, min

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

105

110

115

120

125

130

Inte

nsity

, cps

0.78

0.88

2.05

Figure 9: Chromatogram of Blank Plasma Sample of Clavulanate


1553


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.

https://www.researchgate.net/journal/0974-6943_Journal_of_Pharmacy_Research


1554


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.

Documents

DEVELOPMENT AND VALIDATION OF AMOXICILLIN AND … · History Clavulanic acid is in the form its salts and esters. The acid is a suicide inhibitor of bacterial beta-lactamase enzymes