Journal of Pharmaceurical Rr Biomedical Analysis, Vol. 13, No. 6. pp. 769-776. 199.5 Elsevier Science Ltd Pergamon Printed in Great Britain 073 I -7085/95 $9.50 + 0.00 0731-7085(95)01281-8 Simultaneous determination of the binary mixtures of cefsulodin and clavulanic acid by using first-derivative spectrophotometry J.A. MURILLO,* J.M. LEMUS and L.F. GARCIA Department of Analytical Chemistry and Food Technology, University of Castilla-La Mancha, E-1.?071- Ciudad Real, Spain Abstract: Two-component mixtures of cefsulodin and clavulanic acid were analysed by a first-derivative spectrophoto- metric method using a zero-crossing technique of measurement. The relative ease offered by this derivative technique for the quantification of these drugs, with closely overlapping spectral bands, was clearly demonstrated. As the absorption band of clavulanic acid closely overlaps with that of cefsulodin, both direct and derivative spectrophotometric methods have been investigated and evaluated by an exhaustive statistical analysis of the experimental data. The first-derivative spectrophotometric method was found to be more rapid, accurate and reproducible. The procedure does not require any separation step. The calibration graphs were linear in the range 2.0-56.0 pg ml -’ for cefsulodin and 2.0-28.0 pg ml-’ for clavulanic acid. The lower detection limits of cefsulodin and clavulinic acid (P 0.05 level) were calculated to be 0.16 and 0.24 pg ml-‘, respectively. Mixtures of cefsulodin and clavulanic acid in ratios of 1:4-7:2 were satisfactorily resolved. Both components were also determined in physiological solutions used to prepare intravenous infusions of these antibiotics. Keywords: Derivative spectrophotometry; first-derivative spectrophotometry; cefsulodin; clavulanic acid: simultaneous determination. Introduction reduced sensitivity to ampicillin and enhances the activity of this antibiotic [7]. In con- l3-Lactamase inhibitors are emerging as sequence, the joint administration of these important adjuncts in the chemotherapy of drugs is of clinical interest as is the simul- bacterial infections [l, 21. The use of irrevers- taneous determination of their mixtures. ible enzyme inhibitors, such as clavulanic acid, Clavulanic acid shows an absorption in combination with various p-lactamase- spectrum that totally overlaps that of cef- susceptible p-lactams has significantly sulodin. Derivative spectrophotometry increased the antibacterial spectrum of these presents greater selectivity than does normal drugs [3-51. spectrophotometry and, therefore, offers a Cefsulodin is an almost “pure” antipseudo- convenient solution to the problem of resolving monal cephalosporin and its p-lactam ring can spectral overlap in the analysis of multi- be hydrolysed by same transferable component systems [8, 91. p-lactamases of Gram-negative bacteria [6]. O’Haver [lo] and Fell [ll] demonstrated the Clavulanic acid has large effects on the possibilities offered by derivative spectro- minimum inhibitory concentrations of cef- photometry for the analysis of pharmaceutical sulodin for Gram-negative anaerobes with formulations. Later, this technique has been CHzOH r! I *Author to whom correspondence should be addressed. 769 770 J.A. MURILLO etal. employed for the simulutaneous determination sulodin or 2.0-28.0 kg ml-’ of clavulanic acid of substances of clinical interest, including or their binary mixtures, 5.0 ml of buffer cephalosporins in mixtures, e.g. cephapirin solution (pH 6.8) and water to 25 ml. and cefuroxime [ 121, 7-aminocephalosporanic Absorption spectra were recorded against a acid and 7-aminodesacetoxycephalosporanic reagent blank using a l.O-cm quartz cell. The acid [13]. first derivative was calculated and by measure- The objective of this work was to demon- ment of the signal of the first-derivative spectra strate the ease with which the application of (AA = 8 nm) at 280.0 nm and use of cali- the “zero-crossing” technique to first-deriv- bration graph, the cefsulodin concentration ative spectrophotometry circumvents the was determined. In the same way, the clavu- problem of overlapping spectral bands and lanic acid concentration was determined by allows the simultaneous determination of cef- measurement of the first-derivative signal at sulodin and clavulanic acid in mixtures without 240.0 nm (“zero-crossing” point for cef- the need for prior separation. sulodin) and use of calibration graph. Each calibration graph was constructed varying the concentration of one compound alone within Experimental the established range. Reagents All experiments were performed with Results and Discussion analytical-reagent grade chemicals and water filtered through a Milli-Q (Millipore) filter. A The stability of the aqueous solutions of stock solution containing 200 Fg ml-’ of cef- cefsulodin and clavulanic acid was studied by sulodin (The Sigma Chemical Company) and a recording their absorption spectra. No changes stock solution containing 100 kg ml-’ of in the spectrum of each compound were clavulanic acid (Beecham Pharmaceuticals, observed when the solutions were stored at 4°C BRL 14151) in water were prepared. These in the dark for 7 days. stock solutions were stored protected in the Studies of the influence of pH on the dark and below 4°C. A 0.05 M dihydrogen absorption spectra indicated that the phosphate/hydrogen phosphate buffer solution absorption of cefsulodin and clavulanic acid (pH 6.8) was prepared. was maximum and constant over the pH-range Physiological solutions of 0.9% (w/v) sodium 4.0-8.5. In order to avoid the possible hydro- chloride were supplied by Apiroserum lysis of these compounds in acid and basic (Instituto de Biologia y Sueroterapia, Madrid, media, pH 6.8 was considered to be the Spain) and Grifols (Laboratorios Grifols, optimum; this pH was achieved by adding a Madrid, Spain). A physiological solution of dihydrogen phosphate/hydrogen phosphate 5% anhydrous glucose (dextrose) was supplied buffer. by Apiroserum (Instituto de Biologia y Suero- terapia, Madrid, Spain). 2.5r- Apparatus All spectral measurements and treatment of data were carried out with a Beckman DU-70 spectrophotometer connected to an IBM-PS computer with Beckman Data Leader software [14] and an Epson FX-850 printer. Among other possibilities, this system provides the capability of data manipulation for smoothing data and for generating derivatives. Moreover, special “zoom in/out” and “trace functions” allow the detailed analysis of data and inter- 0.0 / I polation values. 200 220 240 260 280 300 3 WAVELENGTH (nm) Procedure Figure 1 Absorption spectra of cefsulodin (CFS) (28.0 kg ml-‘), Samples were prepared in 25-ml calibrated clavulanic acid (CA) (16.0 pg ml-‘) and a mixture (M); flasks containing 2.0-56.0 kg ml-’ of cef- reference, reagent blank. ANALYSIS OF BINARY MIXTURES 771 Figure 1 shows the absorption spectra of Figure 2 shows the first-derivative spectra of cefsulodin (CFS) (28.0 kg ml-‘), clavulanic cefsulodin (CFS), clavulanic acid (CA) and acid (CA) (16.0 kg ml-‘) and a mixture (M) of their mixture (M). The zero-crossing values of both compounds. It can be seen that cefsulodin cefsulodin appear at 240.0 and 265.0 nm, could be directly determined in the presence of whereas the first-derivative values of the clavulanic acid because its absorption spectrum clavulanic acid are zero at wavelengths greater exhibits a zone where the other component than 264 nm. From these wavelengths, 240.0 does not absorb. However, since the spectral and 280.0 nm were selected as optima to band of clavulanic acid overlaps completely determine cefsulodin and clavulanic acid. with the absorption spectrum of cefsulodin, respectively. there is no spectral feature utilizable to deter- mine clavulanic acid directly. Selection of optimum instrumental conditions The traditional Vierordt method, which The scan speed of the monochromator has involves the use of two simultaneous virtually no effect on the derivative signal equations, and the modified Vierodt method because differentiation is achieved digitally. [ 151 provide results of poor accuracy and The scan speed only determines the distance reproducibility where the absorption spectra of between each of the data points, which are the components are not sufficiently separated. collected every 0.05 s. A scan speed of 300 nm To resolve the problem of closely overlapping min-’ was selected; under these conditions, spectra derivative spectrophotometry, particu- the instrument reads 4.0 data points per nm. larly with digital processing, can be used. This which is equivalent to 0.25 nm between technique involves differentiation of a normal readings. spectrum with respect to the wavelength. The main instrumental parameter affecting The zero-crossing method was used in this the shape of the derivative spectra is the work with satisfactory results. This method wavelength increment over which the deriv- involves the measurement of the absolute atives are obtained (AA). This parameter needs value of the total derivative spectrum at an to be optimized to give a well resolved large abscissa value corresponding to the zero- peak, i.e. to give good selectivity and higher crossing wavelengths of the derivative spectra sensitivity in the determination. Savitzky and of the individual components. Measurements Golay [16] proposed a series of numerical made at the zero-crossing of the derivative tables for the smoothing of experimental data spectrum of one of the two components are a and for the computation of their derivatives. function only of the concentration of the other The main advantage of the method is that component. universal numerical functions are obtained. the convolution of which with the original vector data gives a smoothed vector as well as its successive derivative. Later, Steinier t’t al. [ 171 1 introduced a correction into Savitzky and Golay’s reasoning and a check was performed by using a more general matrician formalism. The Data Leader software has a smoothing algorithm based on the method of Savitzky and Golay, which can result in an improvement in ; the signal-to-noise ratio. Generally, noise decreases with an increase of AA, thus decreasing the fluctuation in a derivative spectrum.