Background: The formulation of sulphamethoxazole (S) and trimethoprin (T) (CO-TRIMOXAZOLE) in a combination mixture is very good pharmacologically since it enhances the efficacy of the individual drugs. However in this combination, difficulties in analysis on ordinary UV spectrophotometry are introduced because the two components give overlapping spectral bands on zero-order. The United States Pharmacopoea (USP)-recommended HPLC analytical method is quite expensive.
Objective: The objective of the present work was to assess whether derivative spectrophotometry could be used to circumvent the overlapping spectral bands of the components and hence use it for routine analysis of the drug.
Study design: Experimental
Methods: The aqueous solution of the individual drugs and their binary mixutres were scanned on zero order and on first derivative at the wave length between 200- 300nm and at the pH of 4.5.
Results: The zero-order spectra of the compounds were completely overlapping. However the first-derivative scan offered better separation and hence T was determined from the absorbance at 237.6nm with negligible contribution from S (since at this point it was reading zero). Likewise S was determined at a wavelength of 259nm when T was reading zero. The linear calibration graphs were obtained for 425mgml^-1 of S and for 4-20mgml^-1 of T.
Conclusion: The method is rapid, simple and can be applied successfully to assay a mixture of the two drugs in pharmaceutical preparations.

Key words: sulphamethoxazole, trimethoprim, derivative spectroscopy, simultaneous determination.

INTRODUCTION

The rationale for the combination of trimethoprim and sulphamethoxazole (CO-TRIMETHOXAZOLE®) for the treatment of infections caused by susceptible bacterial organisms, follows the practical application of the principle that if two drugs act on sequential steps in the pathway of an obligate enzymatic reaction in bacteria, the result of their combination will be supra-additive ¹ . In the co-trimoxazole combination, sulphamethoxazole, a sulphonamide with a structure similar to that of p-aminobenzoic acid (PABA) - a template for folic acid, uses the Woods-Fildes theory of competitive antagonism against PABA, and thus inhibits the incorporation of PABA into folic acid ² . Trimethoprim on the other hand prevents the reduction of dihydrofolate to tetrahydrofolate, which is essential for one carbon transfer reaction crucial for the synthesis of bacterial DNA ³ . While the mammalian cell relies on the ready-made and available folic acid, the synthetic step is necessary for the build-up of the bacterial cell, which assembles its own folic acid. The optimal ratio of the two agents for their synergistic activity has been found to be 5:1 ⁴ . Pharmacologically, this combination has been found useful, and works satisfactorily to reduce both the toxicity of the individual agents and chances for the possible resistance of the organism to the agents, thus enhancing their therapeutic effect. However, analytical difficulties are immediately created since each component of the binary mixture must be ascertained.

At present, the official USP method for the analysis of the combination Sulphamethoxazole–Trimethoprim drug in pharmaceutical preparations commonly utilized, is the High Pressure Liquid Chromatography (HPLC)⁵ , which for a poor country like Uganda, is expensive and hence not appropriate. There are a number of other methods for the analysis, but spectrophotometry seems to offer a very favourable alternative to HPLC.

MATERIALS AND METHODS

Procedure for preparation of standard solutions Standard stock solutions

Sodium acetate-acetic acid buffer (pH 4.5) was prepared by dissolving 6.8g of sodium acetate and 3.0ml of acetic acid (37%), and diluting the solution to 1 litre with water. Suitable spectroscopic settings made were; scan over the range 200nm – 300nm, scan speed, 135nm/min, scanning data interval 0.2nm; and span 50s. Sulphamethoxazole and trimethoprim standard stock solutions (100ugml^-1) were prepared in aqueous ethanolic solution (10%) and kept below 5^oC in a refrigerator for not more than 10 days.

Samples were prepared in 50ml volumetric flasks containing appropriate volumes of the stock solutions. Ten millilitres of pH 4.5 buffer solution, and ethanol (90%) up to 5.5ml were added to the flasks, followed by dilution to mark with water to produce 5-25μgml^-1 of Sulphamethoxazole and 5-20μgml^-1 of Trimethoprim or their binary mixtures.

Five serial dilutions of sulphamethoxazole and trimethoprim, were prepared in triplicate, from the stock solutions as shown in Table 1 in the concentration range of 5-25μgml^-1 and 5-20μgml^-1 respectively. The curves of the solutions were scanned in the range of 200nm – 300nm against aqueous ethanol (10%, pH 4.5) as the blank.

Twenty tablets were accurately weighed and powdered in a mortar. An amount of powder equivalent to the weight of one quarter of a tablet was mixed with 35ml of ethanol in 100ml – calibrated flasks. The mixture was then sonicated for 15 minutes on an ultrasonic bath and subsequently made up to volume with ethanol, filtered through a Whatman No. 1 filter paper and the filtrate used for preparation of the tablet assay solution.

The tablet stock solution buffered to pH 4.5 and ethanol were added to produce an assay solution equivalent to sulphamethoxazole 25μ gml^-1 and trimethoprim 5μgml^-1. The D₁-curve of the solutions (n=5) were obtained and sulphamethoxazole and trimethoprim determined as described above.

In Table 2, the average derivative values produced when the different concentrations of sulphamethoxazole and Trimethaprim were scanned are indicated. Plotting these derivative values against the respective concentrations, the curves yielded the equation D_1-259=0.4505C+0.1943 (correlation coefficient of 0.9996) for sulphamethoxazole and D₁237.6=0.7554C-0.0918 (correlation coefficient of 0.9996) for trimethoprim.

Absorption measurements made by scanning the solution of S, T and the mixture of S and T on zero order are presented. Their overlapping spectral bands are shown in Fig. 1. Derivative spectral bands for the individual components S and T (in serial dilution concentrations) are shown in Figs. 2 and 3 respectively. In Figure 4, first derivative spectra (D₁) are represented by Figures 1, for S, 2 for T and 3 for the mixture. D₁ of the S shows a peak at 259nm, where the dA/dλ , for T is zero, so S can be specifically measured at this wavelength. T on the other hand, has a near trough at 237.6nm where dA/dλ, for S is zero; hence T is exclusively measured at that wavelength. The figure also shows that other zero crossings of the two compounds do appear. However, they appear either at lower wavelengths, where there is possibility of interference absorptions, or are too close for any accurate measurements to be done.

To prove the validity and applicability of the proposed method, synthetic mixtures of different ratios in the concentration ranges stated in Table 2 were assayed. The results were computed against the previously constructed standard curve. Table 3 presents the results of the assay of the mixtures. As can be seen, satisfactory results were obtained with a mean recovery of 100.85±1.28 and 102.70±1.07 for Sulphamethoxazole and Trimethoprim respectively.

As can be seen in Figure 1, absorption measurements of each antimicrobial in a binary mixture appears to be quite impossible because of the total overlap of bands. The band overlap might be solved by Vierordt’s simultaneous equation method¹⁴. However this may be influenced by differences between the sample and reference, or by the matrix in the pharmaceutical formulations leading to erroneous results. Since the mid seventies (1970s), workers like O’Haver ⁶and Fell⁷ found that to resolve the problem of closely overlapping spectra, derivative spectroscopy, with digital processing together with zero crossing, offered the best option. This technique involves the differentiation of the normal spectrum with respect to the wavelength. The first derivative, D₁, of the sulphamethoxazole spectrum shows a peak at 259nm, where the dA/dλ, for trimethoprim is zero, so sulphamethoxazole can be specifically measured at that wavelength. Trimethoprim on the other hand, has a near trough at 237.6nm where dA/ dλ , for sulphamethoxazole is zero; hence trimethoprim is specifically measured at that wavelength.

In the deriative zero crossing method, it is possible to get two or more ‘zero crossings’. In such a case, meticulous assessment of the best crossing point is possible, basing partly on the strength of the signal. Indeed the present work found other ‘zero-crossings’ of the two compounds. However, they appear either at lower wavelengths, where there is possibility of interference absorptions, or are too close for any accurate measurements to be done. In our setting, the smoothing algorithm of the Camspec-Windows software was used to improve the signal to noise ratio of the derivative spectrum. A scanning data interval of 0.2nm was selected. The instrument automatically sets the scan speed at 135nm/s at this data interval. In addition the Camspec windows programme has four smoothing levels and the highest level was found to be the most appropriate. Higher data intervals, that is 0.5nm or 1.0nm gave spectra with less noise in the signals and therefore better higher order spectra compared to the lower data intervals. However these higher orders, that is second, third and fourth-order spectra didn’t give rise to good zero-crossing points for the concentration ranges studied and therefore were not useful in this study.

The linearity of the selected deriative measurements for both antimicrobials was examined. Linear relationships were obtained with the compounds in the concentration ranges cited in Table 2. The stability of ethanolic solutions (10%) of sulphamethoxazole and trimethoprim was studied by recording their absorption spectra. No changes were observed for at least fourteen days when the solutions were stored at 4^oC in the dark. In order to prove the validity and applicability of the proposed method, synthetic mixtures of different ratios in the concentration ranges were assayed. Satisfactory results were obtained with a mean recovery of 100.85±1.28 and 102.70±1.07 for sulphamethoxazole and trimethoprim respectively and this is within the required limits since the USP-acceptable limit is not less than 93.0% and not more than 107.0% stated on the labels⁵.