INTRODUCTION:

Cefuroxime Axetil (CA), (RS)-1-hydroxyethyl(6R,7R)-7-[2-(2-furyl) glyoxyl-amido]-3-(hydroxymethyl)-8-oxo-5-thia-1-azabicyclo[4.2.0]-oct-2-ene-2-carboxylate,7²-(Z)-(O-methyl-oxime),1-acetate 3- carbamate ) is second generation cephalosporin used to treat or prevent infections that are proven or strongly suspected to be caused by bacteria.¹Clavulanic acid administered as potassium salt, is a powerful inhibitor of β-lactamase enzyme and is most often formulated in combination with antibiotics for treatment of infection caused by lactamase producing bacteria.²

Literature survey reveals spectrophotometric method and validated HPTLC methods for CA determination in combination with other drugs.^3-5Stability indicating and bioanalytical chromatographic methods for quantification of CA are also reported.^6-8 Several HPLC methods have been reported for estimation of Potassium clavulanate (PC) in combination with other drugs.^9-13 This paper describes a simple, accurate, precise and sensitive spectrophotometric methods developed for simultaneous determination of Cefuroxime axetil and Potassium clavulanate in the tablet dosage form as no reports regarding this were found in the literature. The proposed methods were optimized and validated in accordance with International Conference on Harmonization (ICH) guidelines.¹⁴

MATERIALS AMD METHODS:

Instrumentation:

The instrument used in the present study was JASCO double beam UV/Visible spectrophotometer (Model UV-550) with slit width fixed at 2 nm. All weighing was done on electronic balance (Model Shimadzu AY -120).

Reagents and Chemicals:

Analytically pure sample of CA was kindly supplied by Maxim Pharmaceuticals Pvt. Ltd. (Pune, MH) and that of PC was supplied by Medriech Pharmaceuticals (Bangalore) used as such without further purification. The pharmaceutical dosage form used in this study was Covatil CV 250 film coated tablets labeled to contain Cefuroxime axetil USP equivalent to Cefuroxime 250 mg and diluted Potassium clavulanate BP equivalent to Clavulanic acid 125 mg per tablet. (Macleods Pharmceuticals, Mumbai India).

EXPERIMENTAL:

Absorbance Correction Method (Method I):

In this method two wavelengths in the zero order spectra (Figure 1) were selected such that one of the drug shows practically nil absorbance at the detection wavelength of the other drug. While other wavelength selected where both the drugs have considerable absorbance. At detection wavelength 277 nm (λ₁), PC has practically nil absorbance so used for direct determination of CA. The other wavelength selected was 218 nm (λ_2, λmax of PC) where PC was estimated after correction for absorbance of CA at this wavelength. The equations obtained for the determination of drug concentrations are:

A₂₇₇

C_CA = -------------- ......(1)

18.204

A₂₁₈ – (38.30 X C_CA )

C_PC = ----------------------------- .....(2)

28.78

Fig. 1: Zero order overlain spectra of CA (10 mcg/mL) and PC (10 mcg/mL)

First Order Derivative Spectroscopic Method (Method II):

This method is based on first order derivative spectroscopy to overcome spectral interference from other drug. First order derivative spectra of both the drugs were recorded with dλ = 4 nm (Figure 2). It was observed that CA showed dA/dλ zero at 230 nm in contrast to PC that has considerable dA/dλ at this wavelength. Further, PC has zero dA/dλ at 300 nm while at this wavelength CA has significant dA/dλ. Therefore these two wavelengths were employed for the estimation of CA and PC without any interference. The calibration curves were plotted at these two wavelengths of concentration against dA/dλ within the above mentioned range. The equations of line obtained to determine concentrations of CA and PC are as follows:

C_CA = (dA/dλ ₃₀₀ – 0.0024) / 0.0012 …. (3)

C_PC = (dA/dλ₂₃₀ – 0.0021) / 0.0012 …. (4)

Fig. 2: First order derivative overlain spectra of CA (10 mcg/mL) and PC (10 mcg/mL)

Preparation of Standard Stock Solutions:

Standard stock solutions were prepared by dissolving separately 10 mg of each drug in 10 mL of methanol to get concentration of 1 mg/mL. 0.1 mL of the stock solution was further diluted to 10 mL with methanol to get a working standard solution of concentration 10 mcg/mL of both CA and PC. Aliquots of working standard solution were further diluted and scanned in the wavelength range of 200-400 nm.

Preparation of Sample Stock Solution:

Twenty tablets were weighed and finely powdered. An accurately weighed tablet powder equivalent to 10 mg of potassium clavulanate was transferred to 10 mL volumetric flask. 10 mL of methanol was added and contents were sonicated for 5 min. The resulting solution was filtered through Whatman filter paper No. 41. Aliquot portion was appropriately diluted with methanol to get required concentration in the linearity range.

Validation of Method:

The method was validated in terms of linearity, accuracy, precision. The linearity of the method was investigated by serially diluting the stock solutions of PC and CA and measured the absorbance at 218 nm and 277 nm for method I and 230 nm and 300 nm for method II. Both the drugs show linearity in the concentration range from 5-50 mcg/mL. To check the accuracy of the method, recovery studies were carried out by addition of standard drug solution to pre-analyzed sample solution at three different levels 50 %, 100 % and 150 %.

RESULTS AND DISCUSSION:

Under experimental conditions described, calibration curve, assay of tablets and recovery studies were performed. Both the drugs obey Beer’s law within the concentration range of 5-50 mcg/mL with high correlation coefficient > 0.998. The proposed method was evaluated by the assay (n = 6) of commercially available tablets containing CA and PC. The results of assay are presented in Table 1. Results of recovery studies are shown in Table 2. The accuracy and reproducibility is evident from the data as results are close to 100 % with low standard deviation.

Table I: Results of commercial formulation analysis

Method

Label Claim (mg/TAB)

% Label Claim

estimated*

(Mean ± S.D)

%R. S. D.

CA-250

PC-125

100.358 ± 1.475

100.644 ± 1.218

1.470

1.210

CA-250

PC-125

99.130 ± 0.166

100.542 ± 0.087

0.829

0.865

* Mean of six determinations, R.S.D. is relative standard deviation

CONCLUSION:

The validated spectrophotometric methods employed here proved to be simple, economical, rapid, precise and accurate. Thus these can be used for routine simultaneous determination of CA and PC in tablet dosage form instead of processing and analyzing each drug separately.

Table II: Recovery studies of CA and PC

Drug	Conc. of drug added		% Recovery (Mean ± S.D)
	% Level	mcg/mL	Method I	Method II
CA	50	10	99.483 ± 2.52 100.098 ± 0.469 101.493 ± 0.163	99.622 ± 1.032 98.644 ± 0.606 100.096 ± 0.269
	100	20
	150	30
PC	50	5	100.076 ±1.012 100.123 ±1.482 101.731 ± 0.208	100.083 ± 0.838 100.291 ± 0.8819 99.527 ± 1.0408
	100	10
	150	15

*Avg. of three determinations

ACKNOWLEDGEMENT:

The authors express their gratitude to Maxim Pharmaceuticals Pvt. Ltd. (Pune, MH) and Medriech Pharmaceuticals (Bangalore) for the gift sample of pure Cefuroxime Axetil and Potassium clavulanate respectively. Special thanks to Dr. K. G. Bothara, Principal, AISSMS College of Pharmacy for providing necessary facilities and his constant support.

REFERENCES:

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