Simultaneous Estimation of Amoxycillin and Potassium Clavulanate in Injection Formulation by RP-HPLC

 

Adarsh Kumar Pathak*, Dr. Akhilesh Gupta

Department of Pharmaceutical Chemistry, School of Pharmaceutical Sciences, Shri Venkateshwara University, Gajraula, Uttar Pradesh, India.

 

ABSTRACT:

Amoxycillin (AMX) and Potassium clavulanate (PTC) are proven for their therapeutic benefits to treats various diseases. AMX and PTC are proven for their therapeutic benefits to treat various diseases. Due to their synergistic actions, a rapid and specific RP-HPLC method was processed and validated as per ICH guidelines. The chromatographic separation was performed with Inertsil 5μ, 250 mm X 4.6 mm, C-18 HPLC column by using phosphate buffer: methanol (80:20) as mobile phase, at 1mL/min of flow rate and 220nm m. The absorption maxima were found to be 246.6 nm for AMX and 257.6 nm for PTC in 0.1M NaOH used as the solvent. The linearity for these drugs at the selected wavelengths lies between 5-200 µg/ml for AMX and 5-200 µg/ml for PTC. Accuracy was determined by recovery study from injection dosages forms were found to be 99.352 for AMX and 99.533 for PTC. Precision of method was determined as repeatability, day to day and analyst to analyst variation and a show the value within acceptable limit i.e. R.S.D was found to be less than 2 percent).

 

 

INTRODUCTION:

Amoxicillin (AMX) is an amino-penicillin (semi-synthetic beta-lactam antibiotic) which widely used against bacterial infections and  gram-positive and gram-negative microorganisms such as actinomycosis, bone and joint infections, biliary-tract infections, gastroenteritis, Lyme disease, gonorrhoea, acute exacerbations of chronic bronchitis, mouth infections, sinusitis, otitis media, pneumonia, typhoid and urinary-tract infections, paratyphoid fever, bacterial meningitis and the prophylaxis of endocarditis.^1-4 The combination of AMX and Potassium clavulanate (PCT) is often available in market.AMX is belongs to the penicillin family member where penicillin nucleus contains of a thiazolidine ring with a β-lactam ring to which is attached a side-chain.

 

This side chain determines most of the pharmacological and antibacterial properties of the penicillin. It kills bacteria by interfering with the synthesis of the bacterial cell wall. Chemically AMX, 7-[2-amino-2-(4-hydroxyphenyl)-acetyl] amino-3, 3-dimethyl-6-oxo-2-thia-5-azabicyclo [3.2.0] heptane-4–carboxylicacidis shown in figure 1. Its spectrum of activity is extended by administration with the β-lactamase inhibitor clavulanic acid.^5-6 Clavulanic acid is a beta-lactam structurally related to the penicillins and produced by the fermentation of Streptomyces Clavuligerus. Clavulanic acid with amoxicillin gives synergistic actions for the skin, treatment of bronchitis and urinary tract, and soft tissue infections caused by beta-lactamase producing organisms. Clavulanic acid inhibits a wide variety of beta-lactamases, commonly found in microorganisms resistant to cephalosporins and penicillins. By inactivating beta-lactamase (the bacterial resistance protein), the accompanying penicillin/cephalosporin drugs may be made more potent. Chemically PTC, (2R,5R,Z)-3-(2-hydroxyethylidene)-7-oxo-4-oxa-1-aza-bicyclo[3.2.0]heptane-2-carboxylic acidis shown in figure2.^7-8 A literature review revealed that many analytical methods such as Spectrophotometric methods,^9-12 reversed-phase high-performance liquid chromatography,^13-15 liquid chromatography column-switching system,¹⁶ solid-phase ion-pair extraction and ultraviolet detection,¹⁷ isocratic liquid chromatographic method with UV,^18-19 LC–UV and LC–MS,²⁰ micellar electro-kinetic capillary chromatography,²¹ thin-Layer chromatography/densitometry,²² gamma radiation induced effects by liquid chromatography on both the individual drugs and their combination²³ methods for the determination of AMX and PTC in the injection dosage form. Hence, the proposed work was under taken with an aim to develop simple, rapid, accurate and economic method for simultaneous analysis of Amoxycillin and Potasium clavulanate in the injection dosage form, using RP-HPLC^24-30 and to validate the developed method^31-34 as per ICH norms.

 

Figure 1 Chemical structure of Amoxicillin

 

Figure 2 Chemical structure of Clavulanic acid

 

MATERIALAND METHOD:

Pure samples of Amoxycillin Sodium and Potassium Clavulanate were gifts from Alkem Pharmaceutical. Methanol (HPLC grade) was obtained from Rankem, India. Monobasic Sodium Phosphate(monobasic) , Phosphoric acid (AR grade) was obtained from Merkindia, India. Water from Milli Q water (YoungLin Basic 370 series) was used throughout the HPLC procedure.

 

Preparation of Standard Stock Solution:

For Amoxycillin Sodium:100mg of AMX was accurately weighed and transferred to a 100ml volumetric flask and dissolution was carried out in diluent (water), (Stock A; 1000mg/ml). Aliquots of stock A were further diluted to get concentration of 25, 50, 100, 150 and 200mg/ml of AMX.

 

For Potassium Clavulanate:-

100mg of PTC was accurately weighed and transferred to a 100ml volumetric flask and dissolution was carried out in diluent (water), (Stock A; 1000mg/ml). 10ml of stock A was diluted   upto 100ml   with diluent (Stock B; 100mg/ml). Aliquots of stock B were further diluted to get concentration of 5,10,20,30 and 40mg/ml of PTC.

 

METHOD DEVELOPMENT:

Study of overlain spectra and selection of wavelengths: -

Stock solutions of 1000µg/ml were prepared for each drug in 0.1M NaOH. From stock solution concentration of 25µg/ml for Amoxycillin and Potassium clavulanate were prepared and scanned over the range of 200-400 nm and over laid spectra were observed. While studying the overlay spectra it was observed that Amoxycillin shows maximum absorbance at 246.8nm while Potassium clavulanate shows maximum absorbance at 257.6nm and selected as l2 and l1 respectively. The absorbances were measured at 256.8nm for Amoxycillin and 246.8 for Potassium clavulanate.

 

Mobile phase selection: -

The scanning of AMX and PTC were done by preparing 100mg/ml solution of both the drugs separately in combination of various solvent systems (varying the ratio and/or nature of organic modifier), at the end of this study Phosphate Buffer: Methanol (80: 20) was selected as the best mobile phase because in that drug was showing good elution. Spectra as follows in figure 3.

 

Figure 3 Chromatogram of 100µg/ml solution of AMX and PTC using Phosphate Buffer (pH-5.0): Methanol (80: 20) as mobile phase

 

Analysis of Laboratory Sample:

The method was checked by analyzing a solution containing known concentration of both drugs (mixed standard). Mixed standard solutions were prepared from standard stock solution as stated in section - 5.2.7 The concentration and results are reported in table 1 and 2.

 

Table 1 Conc. of AMX and PTC in mixed standard

Standard No.	1	2	3	4	5
AMX mg/ml	25	50	100	150	200
PTC mg/ml	5	10	20	30	40

 

Table 2 Result of analysis of laboratory sample

S. No.	Amount present (mg/ml)		Conc. found (mg/ml)		% of amount found
	AMX	PTC	AMX	PTC	AMX	PTC
1	25	5	24.69	4.89	98.76	97.80
2	50	10	49.89	9.82	99.78	98.20
3	100	20	96.82	20.60	96.80	100.30
4	150	30	148.92	31.02	99.28	103.00
5	200	40	201.3	39.96	100.65	99.90
% MEAN					99.128	100.350
S.D.					1.6517	1.9875
R.S.D.					1.666	1.981

 

Analysis of Injection Sample:

Twenty dry injections were taken; average weight was determined and mix well fine powder. Amount equivalent 100mg Amoxycillin and 100mg Potassium clavulanate was taken in 100ml volumetric flask. This was dissolved water and sonicate for 3 min. The volume was made up to mark with water and filtered through whatmann filter paper (no 41).  Filtrate was further diluted with solvent get the final concentration of both drugs on the working range. The responses of final dilutions were observed at selected wavelengths and the concentrations were obtained from regression equation. The procedure was repeated for three times. The result and statistical parameters are recorded in table 3 and 4.

 

Table 3 Analysis of injection formulation

S.No.	Conc.Present(µg/ml)		Conc. Found(µg/ml)		% Conc.Found (µg/ml)
	PTC	AMX	PTC	AMX	PTC	AMX
1	5	25	4.86	25.11	97.2	100.4
2	10	50	9.91	49.97	99.1	99.94
3	20	100	20.03	101.02	100.1	101.02
4	30	150	30.12	149.22	100.4	99.48
5	40	200	39.78	198.36	99.45	99.18

 

Table 4 Statistical data of injection formulation

S. No	Drug	Mean	S.D.	RSD
1	AMX	99.76	1.210	1.27
2	PTC	99.90	1.686	1.70

 

Validation of the Method:

Linearity and Range:  

A series of dilutions were prepared in the concentration range of 5-200mg/ml and calibration curves of AMX and PTCwere plotted between concentration Vs response of and slope, intercept and correlation coefficient value (r²) was determined. The slope, intercept and correlation coefficient value (r²) for both drugs are given in table 5.

 

Figure 4 A UV Spectra of 25µg solution of Amoxycillin in water

 

Figure 5 A Spectra of 25µg solution of Potassium clavulanate in Water

 

Accuracy:  

To test accuracy, recovery studies were performed. To a pre-analyzed sample solution, a definite concentration of mixed standard drugs were added, then the solution was analyzed in the same manner as the laboratory sample and injection samples were analyzed and the recovery was studied. It was repeated for three times to emphasize validation. The % Mean, S.D. and R.S.D of both drugs are given in table 5.

 

Precision:

The repeatability study of the drug was performed for three times in concentration range of 25-100mg/ml for AMX and 5-40 for PTC. The intermediate precision was performed by doing day-to-day variation and analyst- to-analyst variation. The statistical data for both drugs are given in table 5.

 

Repeatability: -

As per section 5.2.7 standard dilutions were prepared and six replicates of each dilution were analyzed in same day for repeatability and results were subjected to statistical analysis. (Table 5)

 

Robustness:  

As per ICH norms, small, but deliberate variations, by altering the pH and/or concentration of the mobile phase were made to check the method’s capacity to remain unaffected. The change was made in the ratio of mobile phase, instead of Methanol: Phosphate Buffer pH 5.0 (80: 20 V/V), Methanol: Phosphate Buffer pH 5.0 (92: 8 V/V), Methanol: Phosphate Buffer pH 5.0 (80: 20 V/V) was used as a mobile phase. (Table 5)

 

Table 5 Validation parameters

METHOD	PARAMETER	AMX	PTC
Linearity	Slope	361.33	42.22
	Y-INTERCEPT	361.33	42.22
	r 2	0.9998	0.9997
	LOD	0.0136	0.130
	LOQ	0.412	0.395
	Robustness	Robust	Robust
	Theoretical plates	3189.33	7525.83
Accuracy	% Recovered	99.352	99.533
	S.D.	1.5662	1.0852
	R.S.D	1.576	1.193
Precision
Repeatability	S.D.	0.7011	0.9753
	R.S.D	0.710	1.000
Day to day	S.D.	0.4303	0.9906
	R.S.D	0.432	0.988
Analyst to analyst	S.D.	0.5612	1.5469
	R.S.D	0.561	1.546

 

RESULTS AND DISCUSSION:

A sensitive, selective, precise and accurate high performance liquid chromatographic method for analysis of AMX and PTC are proven for their therapeutic benefits to treat various diseases. Due to their synergistic actions, a rapid and specific RP-HPLC method was processed and validated as per ICH guidelines. The chromatographic separation was performed with Inertsil 5μ, 250mm X 4.6mm, C-18 HPLC column by using phosphate buffer: methanol (80:20) as mobile phase, at 1mL/min of flow rate and 220nm m. The absorption maxima were found to be 246.6nm for AMX and 257.6 nm for PTC in 0.1M NaOH used as the solvent. The linearity for these drugs at the selected wavelengths lies between 5-200µg/ml for AMX and 5-200µg/ml for PTC. Accuracy was determined by recovery study from injection dosages forms were found to be 99.352 for AMX and 99.533 for PTC. Precision of method was determined as repeatability, day to day and analyst to analyst variation and a show the value within acceptable limit i.e. R.S.D was found to be less than 2 percent).

 

CONCLUSION:

In the research work done, a successful attempt was made for simultaneous estimation of Amoxycillin Sodium and Potassium Clavulanate in injection formulation by reverse phase high performing liquid chromatographic (RP-HPLC) method which was developed by experimentation based on thorough literature survey and ascertained by statistical parameter of sampling. The simplicity, rapidity, reproducibility and economy of the proposed methods completely fulfill the objective of this research work.In the present work, application of RP-HPLC for analysis of the selected drug formulation was successfully attempted using Younglin-900 liquid chromatographic system. The commercial formulations selected for analysis were Clavum-150. HPLC determination of the AMX and PTC were carried out by maintaining various chromatographic conditions throughout the method. The validation results of above developed methods showed that HPLC method is accurate, precise and sensitive with less error, and RSD were found to be less than two which was within the acceptable limit.

 

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Received on 01.07.2021           Modified on 23.02.2022

Accepted on 17.05.2022   ©Asian Pharma Press All Right Reserved