Anal. Chem. 1984, 56, 2339-2341
2339
Difference Spectrophotometric Determination of Efrotomycin in Feed Premixes David W. Fink* and John D. Stong
Merck Sharp & Dohme Research Laboratories, P.O. Box 2000, Rahway, New Jersey 07065
An analytical application to feed premlxes of the red shift in the absorption spectrum of efrotomycin which accompanies the chelation of aluminum ion by this ligand is described. The antibiotic is extracted from the premix sample into ethyl acetate, an aliquot of the extract is charged to a silica adsorption column, and extraneous interferences are removed by further treatment of the column with the same solvent. The anaiyte Is then eluted with methyl alcohol and determined by difference spectrophotometry on the basis of its complexation with aluminum ion. Thls method has an accuracy of f0.5% mean relative error and a precision of f3.5% relative standard deviation (n = 13) over the concentration range 0.1-1.0% efrotomycin in the premix.
The chelation of aluminum ion by the antibiotic efrotomycin has been reported recently (1). That report described the red shift in the absorption spectrum which accompanies chelation. This N-methylhydroxypyridone glycoside (2) contains two tautomeric 0-dicarbonyl units and thus can coordinate as a bidentate ligand via two oxygen donors in analogy to acetylacetone. In the previous report, one representative set of analytical conditions was established to illustrate the use of this spectral shift in a direct spectrophotometric determination of the antibiotic. In that report, the linearity and precision of the Beer’s law calibration were documented. This correspondence describes a specific analytical application to feed premix samples of the chelation of aluminum by efrotomycin. I t is based upon the use of this reaction in a difference spectrophotometric technique, combined with an extraction and separation scheme for application to these samples. In addition, this general technique can be extended to other members of this new class of antibioticsLe., goldinomycin (3), mocimycin (4), and heneicomycin (5). A feed premix is a preparation of the drug on a suitable carrier, diluent, or adsorbent which is diluted further at a feed mill or added directly to a feed for administration to animals (6, 7). Because efrotomycin is biologically active when orally absorbed, the commercial product under development for this antibiotic is a feed premix. In the present method, efrotomycin is extracted from, the premix sample at room temperature into ethyl acetate, and, after centrifugation, an aliquot of the extract is charged to a silica adsorption column. Extraneous interferences are removed by further treatment of the column with the same solvent. Efrotomycin is then eluted with methyl alcohol and determined by difference spectrophotometry based upon ita complexation with aluminum ion. EXPERIMENTAL SECTION Apparatus and Reagents. Absorption spectra were recorded on a Hewlett-Packard Model 8450A diode array spectrophotometer using 1.00-cm quartz cells, and reactions were run in a constant-temperature water bath (Organomation Model 104). The primary standard efrotomycin (Merck Sharp & Dohme Research Laboratories) was stored in the freezer. Anal. Calcd C, 59.78; H, 7.57; N, 2.36. Found: C, 59.69; H, 7,78; N, 2.24. Other (less pure) secondary standard batches of this fermentation-product 0003-2700/84/0356-2339$01.50/0
antibiotic were standardized with reference to this primary reference by reversed-phase HPLC on a Zorbax ODS (Du Pont) stationary phase using a mixed solvent mobile phase of 7030 (v/v) M aqueous H3P04and a detection X of methyl alcohol:8 X 320 nm. Methyl alcohol and ethyl acetate (HPLC grade) were obtained from MCB. Bond Elut silica solid-phase extraction columns were purchased from Analytichem International. Reagent grade anhydrous aluminum chloride (J. T. Baker) was used without further purification. The reagent solution, prepared by dissolving 0.200 g of A1C13 in 100 mL of methyl alcohol, is stable for at least 3 months. Preparation of Analytical Calibration, A stock solution of known concentration was prepared to contain 0.720 mg/mL of efrotomycin by dissolving 0.120 g of the antibiotic in 5.00 mL of methyl alcohol and diluting a 3.00-mL aliquot of this solution to 100.0 mL with ethyl acetate. Further dilutions of this stock solution with ethyl acetate were used to furnish known analyte solutions over the concentration range 30-200 bg/mL efrotomycin. A 5.00-mL aliquot of each calibration standard was charged to a Bond Elut silica adsorption column. Each column is prepared by elution with 1mL of methyl alcohol followed by 2 mL of ethyl acetate in advance of application of the sample. After the sample has been adsorbed on the column, interferences are removed with a 4-mL volume of EtOAc and the drug is then eluted into a 10.00-mL volumetric flask with 4 mL of methyl alcohol. Each flask is then made to volume with methyl alcohol. Two 3.00-mL aliquota of each analytical solution are transferred to separate 50-mL centrifuge tubes. To the portion to be used in the reference cell (i.e., uncomplexed free anal@) is added 1.00 mL of methyl alcohol; to the centrifuge tube containing the solution for use in the sample compartment is added 1.00 mL of AlC13reagent solution. The centrifuge tubes are sealed, and the reaction mixture is incubated in a constant-temperature water bath at 40 O C for 0.5 h to effect complexation. The difference spectrum is then recorded over the wavelength range 2513-450 nm with the chelate solution in the sample cell against the same concentration of uncomplexed free efrotomycin in the reference and at the cell. Absorbance is measured at the 402-nm A, - A3* 322-nm A-, and the difference in absorbance PA = Ais plotted as a function of efrotomycin concentration to provide the analytical calibration line. Sample Preparation. A 2.00-g sample of a feed premix containing efrotomycin in the concentration range 0.1-1.0% is weighed into a 50-mL centrifuge tube and extracted by shaking the sample at room temperature for 30 min with 20.0 mL of ethyl acetate. After centrifugation, a 5.00-mL aliquot of this ethyl acetate extract is diluted to 25.00 mL with the same solvent, and a 5.00-mL portion of this analytical solution is charged to a Bond Elut silica column which has been prepared as described above. The sample is further treated exactly as the analytical reference standard solutions. Concentration is determined from the PA of the sample (A40znrn- A32znrn) with reference to the calibration line. RESULTS AND DISCUSSION Difference Spectrophotometry between the Chelate and the Ligand. In the difference spectrophotometric analytical technique (81, the absorption spectrum of the analyte-aluminum chromophore is measured in the sample cell against the absorption spectrum of the free uncomplexed antibiotic a t the same concentration in the reference cell. Hence, this technique is useful for the analysis of formulations because it compensates for most interferences in the sample 0 1984 Arnerlcan Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 56, NO. 13, NOVEMBER 1984 I
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Figure 1. Difference spectrum of efrotomycin-aluminum complex as a function of analyte concentration. The concentrations in both reference and sample cells in these solutions are 9.5 X 10" M, 1.9,2.8, 3.8,4.7,7.1,and 9.5 X 10" M, and 1.2 X lO-'M.
by having the same interferences present at equal concentration in both the sample and reference cells; hence, these absorbances will cancel. The difference in absorbance, then, which is a direct function of analyte concentration, is independent of other interfering chromophores in the samples which do not exhibit a spectral shift with the analyte perturbation. Because of this property of the technique, the need for quantitative isolation of the analyte is obviated. Figure 1 presents a series of difference calibration spectra over the concentration range 9.5 X 10*-1.2 X M efrotomycin. These spectra are characterized by a ,A, at 402 nm, a Amin a t 322 nm, and isosbestic points at 349 and 284 nm. That these isosbestic points appear at A = O.OO0 indicates the absence of interferences which absorb at these wavelengths. Absorbance is
