DEVELOPMENT AND VALIDATION OF FIRST ORDER DERIVATIVE METHOD FOR SIMULTANEOUS ESTIMATION OF CEFIXIME TRIHYDRATE AND LINEZOLID IN ITS COMBINED TABLET DOSAGE FORM

 

ABOUT AUTHORS:
Chetana Ribadiya*1, Hemang Ribadia2, Nimish Talaviya3, Chandani Joshi3, Ashok Parmar3
1M.Pharm, Smt. R. B. Patel Mahila Pharmacy College, Atkot
2M.Pharm, Drug Regulatory Affairs Department of Pharmaceutical Science, Saurashtra University
3M.Pharm, Smt. R. D. Gardi B. Pharmacy College, Rajkot
*chetana.ribadiya@gmail.com

ABSTRACT
A simple, precise, accurate and reproducible spectrophotometric method has been developed for simultaneous estimation of Cefixime Trihydrate (CEF) and Linezolid (LNZ) by employing first order derivative zero crossing method in Methanol. The first order derivative absorption at 290 nm (zero cross point of CEF) was used for quantification of Linezolid and 228 nm (zero cross point of LNZ) for quantification of Cefixime Trihydrate. The linearity was established over the concentration range of 2-18 µg/ml and 7-15 µg/ml for Cefixime and Linezolid with correlation coefficient r2 0.9970 and 0.9982, respectively. The mean % recoveries were found to be in the range of 98.36 % – 99.45 % and 100.10 % – 101.66 % for Cefixime and Linezolid, respectively. The proposed method has been validated as per ICH guideline and successfully applied to the estimation of Cefixime and Linezolid in bulk and in market formulation.

REFERENCE ID: PHARMATUTOR-ART-1919

INTRODUCTION
Cefixime Trihydrate (CEF) is an oral third generation cephalosporin antibiotic. Cefixime (C16H15N5O7S2, 3H2O), chemically, it is (6R,7R)-7-{[2-(2-amino-1,3- thiazol-4-yl)-2-(carboxymethoxyimino) acetyl]amino}-3- ethenyl-8 oxo-5-thia-1 azabicyclo - [4.2.0]oct-2-ene-2-carboxylic acid trihydrate[1], clinically used in the treatment of susceptible infections including gonorrhea, otitis media, pharyngitis, lower respiratory-tract infections such as bronchitis, and urinarytract infections [2,3].

Linezolid (LNZ) is a synthetic antibacterial agent of the oxazolidinone class of antibiotics. Linezolid is chemically N-{[(5S)-3-[3-fluoro-4- (morpholin-4-yl) phenyl]-2-oxo-1, 3- oxazolidin-5-yl] methyl} acetamide [4]. Clinically used for the treatment of infections caused by multi-resistant bacteria including streptococcus and methicillin resistant Staphylococcus aureus (MRSA). The drug works by inhibiting the initiation of bacterial protein synthesis [2,3]. Both the drugs are marketed as combined dose tablet formulation in the ratio of 200:600 mg CEF: LNZ. Literature survey reveals that Cefixime Trihydrate can be estimated by Spectrophotometrically [5], and by HPLC[6-8]  individually or with other drugs in bulk drugs and in human plasma, while Linezolid can be estimated by Spectrophotometrically [9-10], HPLC [11] in combination with other drugs. However, there is no analytical method reported for the estimation of CEF and LNZ in a combined dosage formulation. Present work describes RP-HPLC method for simultaneous estimation of CEF and LNZ in tablet formulation.

The chemical structures of Cefixime trihydrate(A) and Linezolid(B) are shown in Figure 1. [1, 4]


Figure-1: Chemical structure of (A) Cefixime Trihydrate and (B) Linezolid

MATERIALS AND METHODS

Instrumentation
Double  beam  UV-visible  spectrophotometer (heλios Alpha, Model - V 7.09)    having  two  matched  quartz  cells  with  1  cm  light  path. An Electronic analytical balance (Contech, CA34 Model) was used in the study. 

Material and reagent
Reference standard of Cefixime Trihydrate (gift sample from Sunrise Remedis Pvt Ltd,

Ahmadabad, Gujarat, India) and Linezolid (gift sample from Alembic Pharmaceuticals Ltd.,Gujarat, India). Tablet Zifi-turbo (F.D.C spectrahealthcare Pharma, India) (Label claim: Cefixime 200 mg and Linezolid 600 mg) was used.  HPLC grade methanol from Finar chemicals, Ahmadabad. All other chemicals and reagents used were of AR grade. Mili Q water was use for this study.

Preparation of Standard Stock solution of CEF and LNZ:
Accurately weighed quantity 100 mg of CEF and LNZ were transferred into separate 100 ml volumetric flask, dissolved and diluted up to mark with methanol (100 ml). This will give a stock solution having strength of 1000 μg/ml of each.

Preparation of Working Standard Solution of CEF and LNZ:
100 µg/ml of CEF and LNZ solution were prepared by diluting 1 ml of stock solution to 10 ml with Methanol in separate 10 ml volumetric flask. Suitable aliquots of this solution were diluted up to the mark with methanol to get the concentration range of 2, 6, 10, 14 and 18 μg/ml for CEF and 7, 9, 11, 13 and 15 μg/ml for LNZ.

Selection of analytical wavelength:
2-18 µg/ml solutions of CEF and 7-15 µg/ml solutions of LNZ were  prepared in  Methanol by appropriate dilution of working standard solution and spectrum was recorded between 200-400 nm and all zero order spectrums (D0) were converted to first derivative  spectrum  (D1) using delta lambda 2.0 and scaling factor 20. The overlain first derivative spectrums of CEF and LNZ at different concentration were recorded. The zero crossing point (ZCP) of CEF was found to be 290 nm (Figure 2) and ZCP of LNZ was found to be 228 nm (Figure 3).

Preparation of calibration curve:
Standard solutions of CEF in the concentration range of 2 to 18 μg/ml obtained by transferring (0.2, 0.6,  1.0,  1.4  and  1.8  ml) of CEF working standard solution (100 μg/ml) to the series of 10 ml volumetric flasks and standard solutions of LNZ in the concentration range of 7 to 15 μg/ml were obtained by transferring (0.7,0.9, 1.1, 1.3 and1.5 ml) of LNZ working standard solution (100 μg/ml) to the series of  10  ml  volumetric  flasks. Then volume was adjusted up-to mark with Methanol. All dilutions were scanned in wavelength range of 200 nm to 400 nm. All zero order spectrums (D0) were converted to first derivative spectrum (D1). The absorbance was plotted against the respective concentrations to obtain the calibration curves.

METHODOLOGY:
First order derivative method uses the Zero crossing point (ZCP) of spectra. If ZCP is more than one than we should have to consider ZCP which gives best correlation coefficient. From the overlain spectra of both drugs (as shown in figure 2 and 3), it shows that CEF and LNZ having ZCP at 290 nm and 228 nm respectively. Working  standard  solutions having  concentration  2, 6, 10, 14, and  18  µg/ml  for CEF  and  7, 9, 11, 13 and 15 µg/ml  for LNZ were  prepared and the D1 absorbance at 290 nm (ZCP of CEF) for LNZ and 228 nm (ZCP of LNZ) for CEF  were  measured  and calibrations curve were prepared.

Calibration curve equation for,

CEF: y = 0.0077x + 0.011

LNZ: y = 0.0177x + 0.081

These both equations were used for % recovery of drug from sample solution.

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