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Journal of Drug Delivery and Therapeutics

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Open Access  Full Text Article                                                                                                                                      Research Article 

Development and Validation of a RP-HPLC Method for the Simultaneous estimation of Amoxicillin, Omeprazole and Tinidazole in fixed dose combinations 

Basant Lal*1, Manish Jaimini1, Devesh Kapoor2  

Department of Pharmaceutical Sciences, Maharishi Arvind University, Mundiaramsar, Jaipur, Rajasthan-302041, India

2 Department of Pharmacy, Dr. Dayaram Patel Pharmacy College, Bardoli, Gujarat-394601, India

Article Info:

_________________________________________

Article History:

Received 07 August 2021      

Reviewed 29 September 2021

Accepted 04 October 2021  

Published 15 October 2021  

_________________________________________

Cite this article as: 

Lal B, Jaimini M, Kapoor D, Development and Validation of a RP-HPLC Method for the Simultaneous estimation of Amoxicillin, Omeprazole and Tinidazole in fixed dose combinations, Journal of Drug Delivery and Therapeutics. 2021; 11(5-S):57-62

DOI: http://dx.doi.org/10.22270/jddt.v11i5-S.5020       

_________________________________________

*Address for Correspondence:  

Basant Lal, Research Scholar, Department of Pharmaceutical Sciences, Maharishi Arvind University, Mundiaramsar, Jaipur Rajasthan

Abstract

______________________________________________________________________________________________________

New liquid chromatographic technique was established for the simultaneous estimation of tinidazole, omeprazole and amoxicillin in the fixed dose combination (HP-KIT by Sun Pharma). RP-HPLC elution has performed by using the Phenomenex Luna column (250 mm x 4.6 mm) having internal diameter and the packing material of size 5µm) in isocratic mobile phase of solution A: acetonitrile at a ratio of 80:20 v/v (Solution A consists of Buffer: Acetonitrile: Methanol: Triethylamine in the ratios of 68:22:10:0.01 respectively). The selected flow rate was kept as 1 ml/min and selected wavelength was 230 nm was for detection of the drugs in UV detector. As per the ICH guidelines, the method validation was carried out. Moreover, the different parameters of method such as precision, specificity, linearity, robustness and accuracy were established. The time of retention for the tinidazole, amoxicillin, and omeprazole were 4.021 2.324, and 7.332 minutes respectively. The RP-HPLC approach was robust and accurate, so it is appropriate for repetitive assay of drugs and quality control. This method is effectively used for the assessment of marketable dosage form preparation.

Keywords: RP-HPLC, Amoxicillin, Tinidazole, Omeprazole, Method Development, Method Validation.

 


 

INTRODUCTION: 

Helicobacter pylorus which is a gram-negative bacterium mostly found in the stomach. It was acknowledged in 1982 by Australian scientists Robin Warren and Barry Marshall. The researchers found that this bacterium was present in people suffering from gastric ulcers and chronic gastritis. Its presence was also noticed in patient of stomach cancer and duodenal ulcers. The stomach and duodenal linings was given much harm by the H. pylori harm due to different mechanisms. These ulcers instigated by the H. Pylori are treated by using the different combination of drugs, like proton pump inhibitor (PPIs) drug combined with two antimicrobials drugs, such as amoxicillin and tinidazole.

Chemical Structure of Omeprazole:          

 image

Chemically omeprazole is recognized as 5-methoxy-2-[[(4-methoxy-3, 5- dimethyl-2-pyridinyl) methyl] sulfinyl] benzimidazole. It is formally listed in BP2011 and USPXXXII. Omeprazole is PPIs, mostly employed in treatment of peptic ulcer disorder, ulceration associated with NSAID, in disease related to gastro-esophageal reflux and also in Zollinger-Ellison syndrome. A literature survey shown that estimation of omeprazole performed in pharmaceuticals by using High performance liquid chromatography1-3.

Structure of Tinidazole:  

image

Tinidazole is chemically recognized as 1-(2-ethyl-sulphonyl ethyl)-2- methyl-5-nitroimidazole. It is official published in BP 2011 and USP XXXII. Omeprazole has significant activity against protozoa anaerobic and bacteria. It is used to eradicate H. pylori in peptic ulcer disease with other proton pump inhibitor and antimicrobials. The estimation can be done through HPLC4-6.

Structure of Amoxicillin:    

image

Amoxicillin trihydrate chemically recognized as [2S-[2a,5a,6aˆ(S)]]-6-[[Amino(4-hydroxyphenyl)acetyl]amino]-3,3 dimethyl-7-oxo-4-thia-1-azabicyclo is the most extensively used β-lactam antibiotic to treat  bacterial infections of ear, nose, throat, skin and lower respiratory tract due to susceptible microorganisms. It has significant absorption ability than other β-lactam antibiotics. The marketed formulations of amoxicillin are capsules, suspensions, tablets and injectable solutions. The combination drugs of Amoxicillin enhance the antibacterial effect and bacterial resistance. In literature, numerous analytical techniques have been employed for quantitative determination of amoxicillin by HPLC7-10.

Till date only one RP-HPLC method has been reported by Kasnia et al11 for the simultaneous estimation of these three drugs. This estimation was done for the microsphere formulation. So, it is a prerequisite to create a validated method using RP-HPLC to estimate the three drugs content in tablet/capsule formulation. 

MATERIALS AND METHODS

Instrumentation and Equipment:

There is the establishment of analytical method and validation was done on the HPLC (Make & Model: Agilent 1220 with UV Detector) is equipped with degasser, solvent delivery pump, using Software (Openlab®).

The elution of RP-HPLC was performed employing Phenomenex Luna column (internal diameter of 250 x 4.6 mm and the packing material having size of 5µm) and solution A with isocratic mobile phase: acetonitrile at a ratio of 80:20 v/v (The pH was accustomed to 6.7 by the help of orthophosphoric acid). For detection of the drugs, flow rate was 1 mL/min and UV detector wavelength was fixed at 230 nm.

Reagents and Chemicals

Omeprazole, Tinidazole and amoxicillin were the gifted samples obtained from Uttranchal Research and Testing laboratory, Uttrakhand. Infrared Spectroscopy (IR) and Mass Spectroscopy were employed for the characterization of these samples to examine the purity level.

RANKEM Chemicals has supplied the AR grade chemicals which are orthophosphoric acid (OPA), potassium dihydrogen phosphate, triethylamine, methanol and HPLC grade acetonitrile. Milli-Q water purification system was used for generation of water which was used during analysis (Make & Model: MILLIPORE / Integral 5).

Methods

Chromatographic conditions

Column

Phenomenex Luna C18, column (250 mm x 4.6 mm), 5 µm

Mobile phase

Solution-A: Acetonitrile: Buffer: Methanol: Triethylamine (Ph 6.7) in the ratio of 22:68:10:0.01

Solution A: Acetonitrile (80:20) v/v

Detector

UV detector

Flow rate

1 ml/min

Wavelength

230nm

Injection Volume

20µL

Temperature

35°C

Diluent

Mobile phase

 

Preparation of Standard Solution:

The stock solutions were formulated by dissolving Omeprazole (25gm) in 25 ml flask, followed by addition of 10 ml of diluent and mix well. Make the volume. Take 1 ml of solution from this solution in 50ml flask. Add 31mg of amoxicillin and 25 mg of tinidazole. Mix well with diluent and finally make the volume to prepare concentrations of 20 μg/mL for Omeprazole and 500 μg/mL for Amoxicillin & Tinidazole respectively.

Preparation of Sample Solution:

The sample solutions were formulated by dissolving 1/10 of average weight of drugs in 50ml flask and mix well with diluents to prepare concentrations of 500 μg/mL for Amoxicillin & Tinidazole and 20 μg/mL for Omeprazole respectively.

Method Validation 

As per ICH Q2 guidelines, linearity, suitability, accuracy, specificity, precision, LOD/ LOQ and robustness were considered for validation of developed method12.

Specificity and selectivity

The established method was found to be suitable for omeprazole, amoxicillin and tinidazole, as the blank solution injection has given the confirmation of absence of interfering peak at RT examined substance at 230 nm wavelength. The outcome obtained demonstrates that no interference was exhibited from other material in the established method and therefore it confirms the established method specificity12.

RESULTS

System Suitability 

The test performed for suitability of system is an essential part of method development, were employed to confirm acceptable chromatographic system performance. The evaluation for retention time (RT), peak asymmetry, tailing factor, and theoretical plates (T) were performed. The outcome are summarised below in Table 1


 

 

 

 

Table 1: System suitability parameters of Amoxicillin, tinidazole and omeprazole

Sr. No. 

Property 

Amoxicillin

Tinidazole

Omeprazole

Acceptance criteria 

1. 

Retention Time (RT) 

2.228

4.112

7.256

2. 

Tailing factor (T) 

1.10

1.21

1.01

NMT 2.0 

3. 

Theoretical plates (N) 

6337

4265

3832

NLT 2000 

 

From the above data it was established that that all the suitability parameters of system were within the limit for developed method.

image

Chromatogram: Blank

image

Chromatogram: Standard

 


 

Linearity and Range 

The developed method linearity proves the method ability to deliver an outcome which is directly proportional to analyte concentration in the sample. The amount of amoxicillin, tinidazole and omeprazole were made for linearity in the 80-120% range. The amoxicillin, tinidazole and omeprazole amount in five dissimilar concentrations are 80%, 90%, 100%, 110% and 120% of working standard respectively. The graph was plotted between area of peak and concentrations. The omeprazole, amoxicillin and tinidazole exhibited good correlation coefficients (R2= 0.9992, 0.9982, and 0.9995) and the planned method was linear in 80-120 % concentration range.


 

 

Table 2: Linearity of Amoxicillin, Omeprazole and Tinidazole

S. No.

Compound

Values of X and Y Variables

Correlation co-efficient

1

Amoxicillin

Variable

1

2

3

4

5

0.9982

X

400

450

500

550

600

Y

306529813

336208695

365098652

388299753

415609155

2

Tinidazole

X

400

450

500

550

600

0.9995

Y

114987563

128677481

143320695

158340861

173826962

3

Omeprazole

X

16

18

20

22

24

0.9992

Y

11975684

13659318

15030951

16632982

18237528

Note: X is the concentration of the respective component in μg/mL . Y is the peak response of the respective component in area counts.


 

Linearity Curve

Calibration curve was plotted between peak area and different concentrations. The outcomes were noted for equation of line and correlation co-efficient were also calculated. 

image

image

image

 

Precision

The precision exhibits the closeness between the series of measurements. The developed method precision was established by method precision and system precision. A homogenous sample concentration of 20 μg/mL for Omeprazole and 500 μg/mL for Amoxicillin & Tinidazole were prepared under recommended conditions and determination was performed as well. The outcomes are mentioned in the form of standard deviation (SD) and RSD value. Table 3 and 4 discloses the outcome of method precision and system precision respectively and the developed method is extremely precise as the value for % RSD is less than 2%.


 

 

Table 3: Calculation of %RSD for Amoxicillin, omeprazole and tinidazole (System Precision)

S. No

Compound

No. of Injections

Mean

S.D. 

%RSD 

1

2

3

4

5

6

1

Reference Standard

Amoxicillin

310619562

310645821

310687549

310679852

310705593

310652984

310665227

31474.918

0.010

2

Reference Standard

Tinidazole

145596431

145319566

145748241

146032184

146318547

145524718

145756615

363929.37

0.250

3

Reference Standard

Omeprazole

15715962

15748752

15705487

15774851

15795822

15739856

15746788

34350.517

0.218

 

Table 4: Calculation of %RSD for Amoxicillin, omeprazole and tinidazole and (Method Precision)

S. No

Compound

No. of Injections

Mean

S.D. 

%RSD 

1

2

3

4

5

6

1

Sample

Amoxicillin

365098652

364895162

365132476

365198411

365862325

365710648

365316279

381069.7

0.104

2

Sample

Tinidazole

143320695

143215548

143395682

143486071

143965842

143895243

143546514

311250.46

0.217

3

Sample

Omeprazole

15030951

15025476

15036741

15037245

15040887

15039604

15035151

5846.1862

0.039

Mean represents the average values of six replicates analysis. SD is the standard deviation calculated on the six replicates. RSD is the relative standard deviation.

 


 

Table 5: System Precision and Method precision

Precision 

Drug 

% RSD 

System precision 

Amoxicillin 

0.011

Method precision 

Amoxicillin 

0.106

System precision 

Tinidazole

0.255

Method precision 

Tinidazole

0.221

System precision 

Omeprazole

0.209

Method precision 

Omeprazole

0.045

 

Accuracy

 Accuracy is also known as trueness or recovery. The accuracy was checked by using 80%, 120% and 100% of working strength of amoxicillin, omeprazole and tinidazole. Each level of solution was prepared in duplicate and analysis is done as per the above method. This is usually mentioned in the form of SD and RSD. The outcome expressed that the value of % RSD is less than 2%. The % recovery results are mentioned in Table 6.

Table 6: Summary of assay of Amoxicillin, omeprazole and tinidazole

S. No. 

Level 

Compound

% Average Assay 

%RSD 

1

80%

Amoxicillin

99.49

0.09

Tinidazole

99.83

0.16

Omeprazole

99.12

0.19

2

100%

Amoxicillin

99.96

0.11

Tinidazole

99.50

0.25

Omeprazole

99.26

0.10

3

120%

Amoxicillin

99.04

0.15

Tinidazole

99.01

0.26

Omeprazole

99.80

0.24

 

Assay values of tinidazole were found of in the range of 99.11-99.89, amoxicillin in the range of %99.14-99.86 % and omeprazole in the range of 99.09-99.79%. Moreover, amoxicillin % RSD assay values were in the range of 0.08-0.22 %, tinidazole in the range of 0.15-0.24 % and omeprazole in the range of 0.11-0.25%. The study confirms that the developed method is precise for the estimation of amoxicillin, tinidazole and omeprazole assay over the range of 80-120% of target concentration.

LOD and LOQ (Limit of Detection and Limit of Quantification)

Limit of detection (LOD) and Limit of Quantification (LOQ) exhibits information related to the analyte concentration that yields signal-to-noise around 1 to 10. The serial dilutions are fabricated from the amoxicillin, tinidazole and omeprazole solution for estimation of LOQ and LOD values respectively. The prepared samples were injected into the HPLC system and blank and sample signals were compared for LOD and LOQ calculation. As per the parameters mentioned earlier, LOD and LOQ were calculated for amoxicillin, omeprazole and tinidazole detected values were 9 μg/ml, 10 μg/ml, 27 μg/ml 2 μg/ml and 0.7 μg/ml, 25 μg/ml, respectively.

Robustness 

The robustness of the method was carried out by doing little deliberate changes in the current developed HPLC method process parameters. These parameters include mobile phase flow rate variations, the minute variation in detector wavelength and variation in the proportion of acetonitrile and buffer. Additionally single concentrations of amoxicillin, omeprazole and tinidazole were employed for verification of robustness parameters. The minor variation of parameters can do some noteworthy changes in the RSD values and peak area. This study recapitulated that the developed method is robust under small changes like ± 2 wavelengths, ± 10% flow rate and ± 10% surge and decline in mobile phase and at the diverse column (Inertsil ODS-3, column (250mmx4.6mm), 5 micron. There is no significant change in recovery of omeprazole, amoxicillin and tinidazole. The % RSD values shown in the Table 7 exhibited that insignificant changes were seen after doing the deliberate changes. So, this study describes that the developed method is robust in nature.

Table 7: Robustness Data

Drug 

Parameters 

% RSD 

Amoxicillin, tinidazole and omeprazole

Wavelength minus 

0.003 

Wavelength plus 

0.002 

Flow minus 

0.004 

Flow plus 

0.002 

Mobile phase ratio change 

0.003 

Column Change 

0.001 

Temperature minus 

0.004 

Temperature plus 

0.005 

 

DISCUSSION 

Trial and error method was employed and subsequently a random number of trials with different, mobile phases were employed but the finest separation of Amoxicillin, omeprazole and tinidazole was present in the Solution A: Acetonitrile in the ratio of 20: 80 (in Isocratic mode). Finally, best outcomes were attained with the flow rate programming of chosen mobile phase for the estimation purpose of all the three drugs. Mobile phase was continued for 60 seconds to 15 min with a of 1 ml/min flow rate and detection was done at 230 nm by using UV detector. 

The validation of fabricated and the optimized method of RP-HPLC was done according to the ICH guidelines with reference to the diverse parameters such as limit of detection (LOD) linearity, limit of quantification (LOQ),  accuracy, precision, and specificity. All the results obtained were found in accordance with ICH guideline.

CONCLUSION 

Agilent 1220 LC system was used for the validation reason of the fabricated and optimized liquid chromatographic method for simultaneous assessment of amoxicillin, omeprazole and tinidazole in HP kit combination. The approach for simultaneous determination of tablet/capsule dosage form has not been mentioned earlier. So, the recent method is novel for these drugs determination at a single wavelength which is 230 nm, along with 20μL injection volume and by employing the Phenomenex Luna C18 5μm 4.6*150mm column. The process is very simple, sensitive and fast as well as accurate, precise, linear and robust which is in accordance of guidelines of ICH. The experimentation work demonstrates that the method development of liquid chromatographic approach exhibited good linearity, resolution, and RSD values which is less than 2%, which proves that simultaneous determination of Amoxicillin, omeprazole and tinidazole can be done suitably by this developed method.

ACKNOWLEDGEMENT

Authors are highly thankful to the services provided by the Uttrakhand Research and Testing Laboratory, Uttrakhand.

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