Development and validation of RP-HPLC method for estimation of Cefotaxime sodium in bulk and formulation

 

Esra Tariq Anwer1*, Omji Porwal1*, Rupesh Dudhe2

1Department of Pharmaceutics, Faculty of Pharmacy, Tishk International University, Erbil, KRG/IRAQ. 44001.

2School of Pharmacy, G H Raisoni University, Saikheda, Chhindwara, Madhya Pradesh India 480337.

*Corresponding Author E-mail: esra.bayrakdar@tiu.edu.iq

 

ABSTRACT:

To develop a novel, accurate, precise and linear reverse phase high performance liquid chromatographic (RP-HPLC) methods for quantitative determination of cefotaxime sodium (CFT) in bulk drug and CFT loaded nanoparticles. Different analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD) and limit of quantification (LOQ) were determined according to International Conference on Harmonization ICH Q2B guidelines. The RP-HPLC method was developed by the isocratic technique on a reversed-phase Zorbax C18 (250 × 4.6 mm, 5µm) column with mobile phase consisting of phosphate buffer (pH7.4): acetonitrile (80:20v/v) at flow rate of 1.2 ml/min. Detection was carried out using a UV detector at 254nm. The retention time for CFT was 6.580+0.5min. The standard curve was linear over the concentration range of 10-50μg/ml with r2 close to one (0.999). The limit of detection (LOD) and limit of quantitation (LOQ) obtained for CET were 0.100μg/ml and 0.314μg/ml respectively. The developed and validated method was successfully applied for the quantitative analysis of nanoparticle. The high recovery and low relative standard deviation confirm the suitability of the proposed method for determining the assay and in vitro dissolution of a marketed formulation.

 

KEYWORDS: HPLC, Nanoparticle, Method validation, Quantitative analysis, ICH guidelines.

 

 


INTRODUCTION:

Infectious ailments have always been twisted the threat to humanoid and faunas 1-8. Therefore, the healing is compulsory using suitable antimicrobial mediators. Even though innumerable antibiotics have been established but cephalosporin group of antibiotics are broadly rummage-sale 9. CFT is chemically a sodium 7-[2-(2-amino-4-thiazolyl)-2- methoxyimino acetamido]-3-acetoxymethyl-3-cephem-4-carboxylate (Fig. 1) with molecular formula C16H16N5 NaO7 S2 and it is a third-generation cephalosporin antibiotic10. It Impedes bacterial cell wall blend by binding to one or more of the penicillin- binding proteins (PBPs), which in turn constrains the final transpeptidation step of peptidoglycan synthesis in bacterial cell walls; thus, inhibiting cell wall biosynthesis11.

 

CFT indicated for the treatment of patients infected with Septicaemia (Bacterial infection of blood), Bacterial Meningitis, Bacterial Endocarditis, Bacterial infections of lungs and respiratory tract, Bacterial infections of bones and joints12

 

Literature review revealed that ESR and other RP-HPLC technique13-29, numerous spectrophotometric, TLC and HPLC methods are available for estimation of cefotaxime sodium30-41. But to the simplest of our knowledge there is no such reported HPLC analysis method for estimation of CFT in bulk drug and nanoparticle. In the existing study, we have established a meek, optimized and validated HPLC analysis method for estimation of CFT in bulk drug and nanoparticles. The process was validated as per the international conference on harmonization (ICH) procedures42. This novel validated method has applicability in an industry over and above in academia.

 

Figure 1 Chemical structure of cefotaxime

 

MATERIALS AND METHODS:

Instrumentation:

Liquid chromatographic system from Agilent 1220 Infinity LC comprising of manual injector, Dual gradient pump for constant flow and constant pressure delivery and variable wavelength detector (VWD) connected to Lab Advisor Software for controlling the instrumentation in addition to processing the generated data. Weighing was done on a analytical sensitive balance AT-220 designed by INOVIA IN Istanbul assembled in China.

 

Reagents and chemicals:

Analytically pure sample of CFT was a generous gift from Glenmark Pharma Ltd., Baddi, in the company of their analytical reports. HPLC grade methanol and acetonitrile was obtained from Merck (India) limited. All other chemical used were of analytical grade. Triple distilled water was used for whole experiment was generated in house. CFT loaded nanoparticles was prepared in house.

 

Chromatographic conditions:

The isocratic mobile phase consisted of phosphate buffer (pH 7.4): acetonitrile (80:20v/v), flowing through the column at a endless drift frequency of 1.2 ml/ min. The mobile phase was filtered through nylon 0.22 µm membrane filters and was degassed before use (30 min). A Zorbax (C-18) column (5 µm, 250mm x 4.60mm) was used as the stationary phase. By in view of the chromatographic parameter, sensitivity and selectivity of method for drugs, 254 nm was selected by means of the detection wavelength for UV-Visible detector.

 

Standard preparation:

Standard stock solution

Accurately weighed 10 mg of CFT was transferred into 10 ml volumetric flask, dissolved in 5ml of mobile phase and volume was made up to 10ml with mobile phase to change to concentration of solution 1000 mg/ml (Stock-A), 5ml of stock-A was taken and diluted up to 50ml to become concentration of 100mg/ml (Stock-B).

 

Working standard solution

Working standard solutions were prepared by taking dilutions oscillating from 10-50mg/ml for CFT.

 

Preparation of nanoparticles by double emulsification-solvent evaporation technique

Poly (lactic-co-glycolic) acid nanoparticles containing CFT were prepared by double emulsion-solvent evaporation technique (w/o/w) with some modifications. Briefly, Take 20 mg/ml solution of CFT in phosphate buffer 7.4 and add 5% w/v solution of PLGA in 20 ml chloroform & acetone mixture (in ratio 2:5) under stirring at 1,500 rpm for 30 min and then the mixture was sonicated (Ultrasonic processor Electrolab-210 US) at 80% amplitude for 1 min on ice. Next, first emulsion was added to 10 ml of an aqueous solution of PVC ((3.0% w/v) under sonication for 15 min on ice to form w/o/w double emulsion. Subsequently, the resultant emulsion (w/o/w) was added to 30 ml of an aqueous solution of 0.1% PVC and vigorously stirred overnight at room temperature to evaporate organic solvent. The PLGA NPs were then centrifuged at 15,000 rpm at 4°C for 20 min and washed three times with deionized water followed by lyophilization (Lyomax made in Hyderabad). NPs were stored at 2-8°C for further study.

 

RESULTS AND DISCUSSION:

Chromatography:

The mobile phase was chosen after several trials with methanol, isopropyl alcohol, acetonitrile, water and buffer solutions in various proportions and at different pH values. A mobile phase consisting of phosphate buffer (pH7.4): acetonitrile (80:20v/v) was selected to accomplish maximum separation and sensitivity. Stream rates between 0.5 and 1.5 min were studied. A flow rate of 1.2 ml/min gave an optimal signal-to-noise ratio with a judicious separation time. Using a reversed-phase C18 column, the retention times for CFT was observed to be 6.580+0.5min. Total time of investigation was less than 8 min. The maximum absorption of CFT was detected at 254nm and this wavelength was chosen for the analysis Fig. 2

 

(A)

 

(B)

 

(C)

Figure 2 Chromatograms of (A) Blank mobile phase (B) CFT (15μg/ml) as reference substances (C) CFT (20μg/ml) in formulation

 

System suitability:

System suitability parameters for example number of theoretical plates, HETP and peak tailing are determined. The outcomes obtained are shown in Table 1. The figure of theoretical plates for CFT was 3555.50.

 

Table 1 Results of system suitability parameters

Parameters

CFT

AUC*

515.99

No. of Theoretical Plates

3555.50

Tailing Factor*

1.23

Retention time*

6.580

Calibration range (μg/ml)

10-50

*Each value is the mean ± SD of six determinations

 

Linearity:

The calibration curve was linear over the concentration range of 10-50μg/ml for CFT. The linearity was represented by a linear regression equation as follows:

Y (CFT) = 50.26conc+3.121 (r2 = 0.999)

 

Accuracy:

Recovery studies were performed to calculate the accuracy of developed method to preanalysed sample solution, a fixed concentration of standard drug (80%, 100%, and 120%) was added and at that time its retrieval was analyzed. The value of percentage RSD was found less than 2 (0.104, 0.102 and 0.170) show good recovery at all three level 80, 100 and 120% respectively. Each level was made in triplicate Table 2.

 

 

Table 2 Results of recovery study

% Level

% Mean±SD*

 

CFT

80%

99.61±0.104

100%

99.42±0.102

120%

99.78±0.170

* Value of three replicate and three concentrations

 

Precision:

Repeatability

Five dilutions in three replicates were analyzed in the same day for repeatability and results were found within acceptable limits (RSD < 2) as shown in Table 3.

 

Intermediate precision

Five dilutions in three replicates were analyzed on two different days and by two analysts for day-to-day and analyst-to-analyst variations and results were found within acceptable limits (RSD < 2) as shown in Table 3.

 

Table 3 Statistical data for precision

Statistical parameter

CFT

Mean*

S.D*

R.S.D*

Repeatability

99.43

0.065

0.065

Intermediate Precision

(I) (A day to day)

99.27

0.041

0.383

(II) Analyst to Analyst

99.22

0.166

0.670

*Mean of 15 determinations (three replicates at five concentration level)

 

Detection Limit and Quantitation Limit:

The LOD and LOQ of developed method were calculated based on the standard deviation of response and slope of the linearity curve Table 4.

 

Table 4 LOD and LOQ

Name

LOD (mg/ml)

LOQ (mg/ml)

CFT

0.100

0.314

 

Analysis of (In-house) nanoparticle formulation:

The assay value of drugs was close to 100, SD and % RSD are less than 2 indicate the no interference of excipients in the estimation of drug Table 5.

 

Table 5 Analysis of Prepared nanoparticle

S. No.

Parameter

CFT

1.

Mean

99.81

2.

S. D.

0.106

3.

% RSD

0.106

 

CONCLUSION:

The projected scheme was successfully pragmatic to the fortitude of CFT from bulk and pharmaceutical (In-house) nanoparticle invention. The proposed RP-HPLC method was validated as per the International Conference on Harmonisation (ICH) Q2B Guiding principle and was found to be appropriate for routine quantitative analysis of CFT by HPLC in pharmaceutical dosage form. The fallouts of linearity, precision, accuracy and specificity, were demonstrated to be within the limits. The method makes available selective quantification of CFT with no interference from other formulation excipients. The projected scheme was highly reproducible, reliable, rapid, robust and specific. Consequently, a high proportion of recovery and the run time of less than seven minutes consent its application for the routine determination of CFT in the pharmaceutical dosage form.

 

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Received on 16.09.2021           Modified on 20.12.2021

Accepted on 12.02.2022         © RJPT All right reserved

Research J. Pharm. and Tech. 2022; 15(7):3114-3118.

DOI: 10.52711/0974-360X.2022.00521