Analyst, May, 1968, Vol. 93, $$. 319-322 319 The Determination of Trace Amounts of Methallibure in Pig Meals BY H. E. HUDSON AND B. PEARSON (Imperial Chemical Industries Limited, Pharmaceuticals Division, Hurdsjield Industrial Estate, Maccksjield, Cheshire) A method has been developed for determining trace amounts of methal- libure in pig meals. Because of the difficulties to be overcome in developing a method for this purpose, it was first necessary to isolate the methallibure from the meal constituents, by using column partition chromatography, and to determine its concentration by monitoring the eluent fractions spectro- photometrically. The solvent partition system used also resolves the active agent from its decomposition product, and the method is, therefore, applicable to stability studies.METHALLIBURE (1-cc-methylallyl-6-methyldithiobiurea), I, is a veterinary drug used to synchronise oestrus in pigs; it is marketed for this purpose as a pre-mix (I.C.I. Ltd., “Aimax”), which is subsequently diluted with pig meal. C&= CH.CH.NH.C.NH.NH.C.NH.CH, II S LH, S II I It is readily soluble in methanol and shows an absorption maximum at 250 mp (E:2m = 1250) ; this property can be used for its determination in pre-mixes containing inorganic diluents. The detemination of methallibure in pig meals is more difficult for the following reasons. (1) Simple solvent extraction to remove the active agent from the meal also removes meal constituents and hence produces a high level of background absorption in the ultraviolet spectrum. (2) Methallibure has been shown to oxidise in meals to give 2-a-methylallylamino- 5-methylamino-l,3,4-thiadiazole, 11.I1 This compound has an absorption maximum in methanol at 273 mp (EiZ = 489) and exerts appreciable absorption at 250 mp, the absorption maximum of methallibure itself (see Fig. 1). (3) It has been observed that methallibure is unstable in methanol extracts of meals. This instability is attributed to incompatibility with some methanol-soluble component of the meal, as yet unidentified. Therefore, the present work was undertaken with the object of developing a method of assay specific for methallibure and applicable to a wide range of meals containing an active agent content of between 50 and 100 p.p.m. 0 SAC and the authors.320 0.7 0.6- E m 0.5- v x 2 0'4- aJ U 8 0.3- n U .- - - U 0 0.2- 0.1 HUDSON AND PEARSON: DETERMINATION OF TRACE [ArtfZ&St, VOl.93 - - 'I Wavelength, mp Ultraviolet spectra for : A, meth- allibure; and B, the decomposition product of methallibure in methanol Fig. 1. EXPERIMENTAL For reasons already stated, it is advisable to separate the drug from any degradation product and to remove, or minimise, contamination from solvent-soluble meal constituents. Column-partition chromatography has been used for this purpose, and it has been shown that the active agent can be isolated by direct application of the medicated meal to the column, followed by elution in the normal way. Under these conditions, the bulk of the eluent-soluble meal constituents leave the column in the first three fractions, but trace amounts influence the first absorption minimum of the methallibure chromatogram (see Calculation of results).There is no indication that methallibure is unstable in the partition - solvent system used. By using the procedure described below, evidence was obtained for the isolation of the methallibure from its oxidation product and is presented in Fig. 2, which shows the characteristic chromatogram for the active agent obtained from a sample of methallibure to which has been added 50 per cent. of its oxidation product. Confirmation of the presence of methallibure was obtained from the ultraviolet spectrum of the eluent in fractions 32, 34 and 42 (Fig. 3). I I I I l l I 10 20 30 40 50 Fraction number Wavelength, mp Fig. 2. Evidence for the isolation of methallibure from its oxidation product Fig.3. Methallibure contaminated with 50 per cent. of its decomposition product: A, fraction 34; B, fraction 32; and C, fraction 42 METHOD APPARATUS- fitted with a sintered disc and tap, was used. Chyomatographic column-A column of 700-mm length and 20 to 23-mm internal diameter,May, 19681 AMOUNTS OF METHALLIBURE IN PIG MEALS 321 REAGENTS- Acid-washed Celite-Transfer 500 g of Celite 545" into a 3-litre beaker and add 2 litres of concentrated hydrochloric acid. Stir the mixture to an even paste and allow it to stand, with periodical stirring, during 12 hours. Decant the bulk of the acid and suspend the residue in 1 litre of water. Filter through a Buchner funnel and wash the residue with water until free from acid.Continue the washing with 500 ml of methanol, and finally wash with 1 litre of a mixture of equal parts of analytical-reagent grade methanol and ethyl acetate. Dry the residue at about 100" C until free from solvent odour. Hexalze-The optical density of the hexane, when read in a 1-cm cell at 254 mp against a 1-cm cell air blank, must not exceed 0.7. If the optical density reading is greater than 0.7 the hexane must be purified as described below. Transfer 10 litres of hexane and 100ml of oleum (20 per cent. sulphur trioxide) into a suitable container and stir rapidly with a stainless-steel stirrer for 30 minutes. Separate the oleum and add an excess of crushed ice. Wash the hexane successively with two 1-litre volumes of water, two 1-litre volumes of 5 per cent.sodium hydrogen carbonate solution and two 5-litre volumes of water. Dry the washed hexane over anhydrous calcium chloride and distil it, collecting the fraction boiling between 65" and 68" C. SOLVENT SYSTEM- purpose reagent grade formamide and 100 ml of hexane. 5 minutes and allow it to stand for 45 minutes before use. phase and the lower layer the eluent phase. Transfer into a 2-litre separating funnel 900 ml of chloroform B.P., 100 ml of general- Shake the mixture vigorously for The upper layer is the stationary PREPARATION OF STANDARD- Accurately weigh about 30 mg of pure standard methallibure and dissolve it in 100 ml of stationary phase. Add 2.0ml of this solution to 4 g of acid-washed Celite, contained in a 100-ml beaker, and mix well. PREPARATION OF SAMPLE- transfer it into a 100-ml beaker containing 4 g of acid-washed Celite and mix well.Accurately weigh sufficient meal to contain about 0-25 to 0.5mg of active agent and PREPARATION OF CHROMATOGRAPHIC COLUMN AND SUBSEQUENT TREATMENT- Transfer 25 g of acid-washed Celite into a 250-ml beaker and add 12.5 ml of stationary phase with a 20-ml straight-sided pipette. Thoroughly mix the stationary phase with the Celite and lightly pack the mixture into the column, adding the mixture in portions of about 3g, and packing down with a tamper after each addition. Add the dry sample mixture reserved from Preparation of sample to the top of the stationary phase in the column, taking care to carry out this latter operation quantitatively. Open the tap at the bottom of the column and carefully add eluent phase to a depth of about 12 inches above the packing.Continue the development with eluent phase and collect successive 10-ml volumes of eluate in 6 x 1-inch stoppered test-tubes. Collect sixty-two fractions and add to each fraction 3 - O m l of analytical-reagent grade methanol and mix well. Measure the optical density of each fraction in a 1-cm cell on a suitable spectrophotometer at 253 mp (the absorption maxima for methallibure in the eluent phase) against a reference solution consisting of 10 ml of eluent phase and 3.0 ml of analytical-reagent methanol. Repeat the above procedure by using the standard mixture reserved from Preparation of standard in place of the sample. The recommended time for a 10-ml fraction is 12 to 2 minutes.CALCULATION OF RESULTS Methallibure in pig meal produces a non-symmetrical chromatogram (Fig. 4) caused by trace contamination with eluent-soluble meal excipients. The residual absorption is determined by drawing a line to strike the minima of the chromatogram tangentially, and * Johns-Manville Co. Ltd., 20, Albert Embankment, London, S.E.l.322 HUDSON AND PEARSON I I I I 10 20 30 40 50 Fraction number Fraction number Fig. 4. Methallibure in pig meal Fig. 6. Methallibure the absorption minima are read. The average of these two values gives the residual absorp- tion. Then, 2 Optical densities between minima of sample peak - (residual absorption x number of fractions) X Optical densities between minima of standard peak - (residual absorption x number of fractions) x 100 Weight of standard (mg) Weight of sample (mg) = Percentage w/w of methallibure content. RESULTS AND DISCUSSION Samples were chosen to cover a wide variation of meal composition and analysed by the proposed method, after fortification with known amounts of methallibure. Figs. 4 and 6 show typical chromatograms for a medicated meal and for methallibure; the results shown in Table I illustrate quantitative recovery of methallibure. The proposed method gives results well within the accuracy usually considered acceptable for products of this type. TABLE I RECOVERY EXPERIMENTS ON DIFFERENT PIG MEALS FORTIFIED WITH ADDED METHALLIBURE Methallibure initially present, p.p.m. 100 100 100 100 100 880 880 880 880 Methallibure recovered, p.p.m. 104 100 100 97 97 860 870 840 860 Received November 13th, 1967