Analyst, August, 1973, Vol. 98, @. 593-595 593 Determination of the Antioxidant 1,3,5 -Trimethyl-2,4,6-tri(3’,5’-di- t-butyl-4’- hydroxy- benzy1)benzene in Feeds BY G. F. BORIES (Station Centrale de Nutrition, Centre National de Recherches Zootechniques, 78- Jouy-en- Josas, France) 1,3,5-Trimethy1-2,4,6-tri( 3’, 5’-di-t-butyl-4’-hydroxybenzyl) benzene (Ion- ox 330) is an antioxidant that is used for the preservation of plastic food wrappings. Its use as a fat stabiliser could be extended to animal feeds. By using the method described, 200 p.p.m. of Ionox 330 in a feed can be determined with good reproducibility. The method consists in extracting the Ionox 330 with chloroform, purifying it by using thin-layer chromato- graphy, and measuring at 522 nm the colour developed in the presence of iron (111) chloride and 2,2’-bipyridyl.Possible interference from 2,6-di-t- butyl-p-cresol (butylated hydroxytoluene; BHT) can be avoided by using this chromatographic technique. THE use of 1,3,5-trimetliyl-2,4,6-tri(3’,5’-di-t-butyl-4’-hydroxyb~nzyl)benzene (Ionox 330”) is authorised in France and several other countries as a stabiliser for the plastic wrappings that are used for packing foods. This substance, which has a high relative molecular mass, is not absorbed in the intestine, thus making it attractive as an antioxidant for stabilisation of fat in animal feeds. A method for determining it in lard, which involves the use of gas- liquid chromatography, has been proposed.1 The high boiling-point of this substance (above 300 “C) presents severe drawbacks, especially the need to heat the chromatographic column to 330 “C. Consequently, the column gives uncertain reproducibility and has a limited life because of fats carried over during the extraction.In order to avoid these difficulties, we tried a colorimetric method of determination that involved the use of Emmerie and Engel’s non-specific reaction2 after purification by thin-layer chromatography. EXPERIMENTAL EXTRACTION OF IONOX 330 FROM THE FEED- Several solvents were tried, but complete extraction could be achieved only by boiling the feed with chloroform under reflux. An aliquot of the extract was concentrated to one tenth of its volume by evaporation in a vacuum before being purified by thin-layer chromato- graphy on silica gel. In order to prevent oxidation of Ionox 330 during the extraction and concentration steps, hydroquinone was added to the feed just before extraction.If this precaution is not taken, as much as 20 per cent. of it may be destroyed in several hours. PURIFICATION OF THE EXTRACT- The concentrated extract is purified by thin-layer chromatography under the following conditions: chromatography on silica gel activated for 30 minutes at 105 “C; elution with cyclohexane - toluene mixture (3 + 2); and simultaneous chromatography of some of the same extract so as to produce indicator spots, which are rendered visible by spraying them with potassium permanganate or antimony(V) chloride solution. The corresponding area of unsprayed plate is scraped off and extracted with chloroform. OUTLINE OF METHOD OF DETERMINATION- We attempted to find a very sensitive colorimetric method for the determination of Ionox 330 that had been purified by thin-layer chromatography. We adapted Emmerie and Engel’s method, which has been used with success for determining several antioxidants3 and is based on the reduction of iron(II1) chloride to iron(I1) chloride and reaction of the latter with 2,2’-bipyridyl to give a red-coloured complex that absorbs at 522 nm.The reaction is * Shell Chemical Company registered trade name. @ SAC and the author.594 BORES : DETERMINATION OF 1 ,Q,G-TRIMETHYL- [Analyst, Vol. 98 carried out in methanolic solution, the colour development taking 30 minutes, after which time the colour is measured. In order to use the chloroform extract of the silica gel adsorbent directly, we carried out the colour development in chloroform - methanol (1 + 1) for 30 minutes.A graph was drawn representing the colour intensity obtained after 30 minutes for a given Ionox 330 concentration in terms of the value of the methanol to chloroform ratio (Fig. 1). It shows that a small variation in the 1 : 1 value for this ratio results in large variations in the reading, and the ratio must therefore be rigidly controlled in order to obtain consistent results. The 2,2'-bipyridyl - iron(I1) complex has a high specific absorption at 522 nm ( E = 3 x lo4). By plotting optical density against concentration of Ionox 330 at concen- trations from 5 to 15 pg ml-l a calibration graph was prepared, which passed through the origin.By using this graph, 5 pgml-1 of Ionox 330 can be accurately determined and 0.5 pg ml-l detected. 0 1 2 3 4 Methanol to chloroform ratio Fig. 1. Effect of the methanol to chloroform ratio on the intensity of the colour developed after 30 minutes with 10 pg ml-l of Ionox 330 METHOD APPARATUS- were used. REAGENTS- Desaga thin-layer chromatographic apparatus and a Jobin and Yvon spectrophotometer Silica gel-Kieselgel G, Merck. Antimony( V ) chloride solution, 25 per cent. VlV in cyclohexane. Potassium permangunate solution, 1 per cent. MethanoZ-Analytical-reagent grade. 2,2'-Bipyridyl (Eastman Kodak) solution, 2 per cent. m/V in metlzafiol-Keep the solution in a cold and dark place. Iron(III) chloride solution, 2 per cent. m/V in methanol. PROCEDURE- Weigh 10 g of a feed containing 200 p.p.m.of Ionox 330 into a 250-ml round-bottomed flask, add 0-5 g of hydroquinone, then extract the mixture by boiling with 100 ml of chloro- form under reflux for 10 minutes. Cool, then filter the mixture on a filter-paper and evaporate 50 ml of the extract contained in a flask down to about 1 ml in a vacuum. Transfer the concentrate with a pipette into a 5-ml vial, rinsing the flask several times with small portions of chloroform, and make the volume up to 5 ml. With a microsyringe, place 0.5 ml on a plate (20 x 20 cm) coated with a 0-5 to 0-75-mm thick layer of silica gel and activated at 105 "C for 30 minutes. Arrange margins on each side of the plate that are wide enough to enable a spot of the same extract to be placed on each margin so as to assist in localisingAugust, 19731 2,4,6-TRI(3’,5’-DI-t-BUTYL-4’-HYDROXYBENZYL)BENZENB IN FEEDS 595 the site of the Ionox 330 after chromatography.Place the plate in a tank containing the cyclohexane - toluene mixture (3 + 2). After migration of the spots is complete (30 to 80 minutes), dry and render the two spots in the margins visible by spraying with either the antimony(V) chloride or permanganate solution, protecting the centre of the plate by covering it with a glass plate. Outline and scrape the area of the silica gel strip containing the anti- oxidant, and transfer the powder into a small chromatographic column. Elute the column with chloroform and collect the eluate in a 10-ml graduated tube (or vial), ensuring that the volume of eluate does not exceed 5 ml (which is sufficient for complete elution of the Ionox 330), then adjust the volume to exactly 5 ml with chloroform (this constitutes tube X); 5 ml of chloroform in a 10-ml tube (or vial) constitutes the blank solution (tube T).A 5-ml volume of chloroform containing 50 pg of Ionox 330 and another 5-rnl volume containing 100 pg of Ionox 330, placed in two 10-ml tubes, constitute the standard range (tubes A and B, respectively). In subdued light, add to each of the four tubes A, B, X and T, 1 ml of iron(II1) chloride solution and 1 ml of 2,2’-bipyridyl solution; shake the tubes, then complete the volumes to LO ml with methanol. Allow the colour to develop for 30 minutes, then read the absorption at 522 nm as rapidly as possible, with tube T as a reference.RESULTS AND DISCUSSION The method described has been used for the determination of Ionox 330 contained in several types of feeds. The product was incorporated in the proportion of 200 p.p.m. To test the reproducibility of the method, a series of determinations was performed on a control feed to which were added, just before extraction, 200 p.p.m. of Ionox 330 dissolved in a small volume of chloroform. The results shown in Table I were obtained from ten successive determinations on each of the feeds. TABLE I RESULTS FOR RECOVERY OF IONOX 330 FROM FEEDS Ionox found in control rat feed with 200 p.p.m. added* 192 184 178 184 182 186 194 184 182 182 Average . . .. . . 184.8 Average recovery, per cent. 92.5 Coefficient of variation . . 2.6 Ionox 330 found in feeds stated to contain 200 p.p.m.of this additive, p.p.m. Rat feed Highly pigmented - for laying hens Batch I Batch I1 (lucerne meal) 200 200 192 216 191 216 203 189 184 200 176 182 210 190 186 200 186 176 196 178 194 190 178 204 186 178 184 196 194 180 199.7 186 189-8 100 93 95 4.4 4.4 6.4 I Pig feed 196 192 212 178 184 180 206 200 188 182 192 96 5.6 * Added in solution. The recovery rate of the additive is satisfactory. It should be noted that the presence of pigments (from lucerne meal) does not affect the percentage recovery. A good separation of Ionox 330 from interfering substances, as well as from 2,6-di-t-butyl-~-cresol (butylated hydroxytoluene; BHT), can be obtained by using the thin-layer chromatographic method described. Finally, by making use of thicker silica gel layers with possibly several elutions with the same solvent system, it should be possible to chromatograph larger amounts of the extract and thus determine 50 p.p.m. of Ionox 330 in a feed with equal precision. We are grateful to the Compagnie de Produits Chimiques Shell (Paris) for permission to publish this paper, and we thank Mrs. E. Biette for technical assistance. REFERENCES 1. 2. 3. “Determination of Ionox 330 in Lard,” Shell Chemical Company, unpublished report. Emmerie, A., and Engel, C., Red Trav. Chim. Pays-Bas Belg., 1938, 57, 1351. Mahon, J. H., and Chapman, R. A., Analyt. Chew., 1951, 23, 1116.