(6) Harding, J. A . , Zbzd., 54, 1254 (1961). (7) Herbst, J. H. E., Can. J . Chem. 30, 668 (1952). (8) Kenaga. E. E., Doty, A. E., Hardy. J. L.. J . Econ. Entomol. 55, 466 (1962). (9) Loudon, J. D., Scott, J. A., J . Chem. Soc. 1953, p. 265. (10) Metcalf, R. L.? Agr. Chem. 16, No. 6, 20 (1961).
ANIMAL METABOLISM
(1 1) Monier-Williams. G. LV.\ J . Chem. Soc. 119, 803 (1921). (12) O'Brien. R. D.. Matthysse. J. G.: .4gr. Chem. 16, S o . 11. 28 (1961). (13) Robinson, H. M. C.. Rathbun, J. C.. Science 127, 1501 (1958). (14) Shorey: H. H., H. T.> .Anderson: L. D.. J . E m . EntOmOl. 55, 5 11962).
(15) Wise, L. E., Murphy, M., D'Addieco, A. A , Paper Trade J . 122, 35 (1946).
Receiued f o r reiiew S o t e m b e r 75, 7963. Accepted March 23. 7964. Division of Agricultural and Food Chemistry, 745th Meeting, ACS, A'ew York, September 7963.
OF I N S E C T I C I D E S ELINOR WILLIAMS, RICHARD W. MEIKLE, and CARL T. REDEMANN Bioproducts Research, The Dow Chemical Co., Walnut Creek, Calif.
Identification of Metabolites of Zectran Insecticide in Dog Urine
The metabolic study of Zectran in dog urine i s described. Free 4-dimethylamino-3,5xylenol and water-soluble conjugates of 4-dimethylamino-3,5-xylenol and 2,6-dimethylhydroquinone are identified as metabolites.
T
EFFECTIVEXESS of Zectran (Registered trade-mark of T h e Dow Chemical Co.), 4-dimethylamino-3,5xylyl methylcarbamate. for the control of mollusks and arthapod pests of plants is \vel1 documented (7). T h e potential \videspread use of this compound raises a problem concerning the manner in Lvhich a n animal can detoxify and eliminate the ingested pesticide from the body. T h e present investigation aims at identifying the metabolites of Zectran in dog urine.
was fed 150 grams of rations containing 20 p.p.m. labeled Zectran twice daily for 7 days. At the end of this time. the labeled Zectran was replaced by inactive material and feeding was continued. Chromatography and Counting Procedures. These procedures are the same as those already described by IVilliams e t . ai. (7). R, values and solvent systems are given in Table I. Fractionation. Urine collections were commenced at the time the feeding of the radioactive material started and continued for 5 days after the feeding ceased, at \vhich time no more radioactivity was present. T h e samples were stored in covered jars a t 4 ' C. T h e urine samples \vere combined and concentrated in vacuo a t - extracted \vith peroxide-free ether 134' C.) for 16 hours. (Peroxidefree ether \%.as prepared by washing U.S.P. ether with a n aqueous suspension
HE
Materials and Methods Feeding. A 5-month old male beagle \vas preconditioned by feeding 6 mg.
( 2 0 p p m . ) of inactive Zectran twice daily in his rations. ivhich consisted of Purina Dog Chow (Ralston-Purina Co., St. Louis. Mo.). T h e inactive Zectran \vas replaced by 4-dimethylamino-3.5xylyl-a". 3-Cl42methylcarbamate, specific activity 0.031 mc. per mmole. T h e dog
Table 1.
R i Values of Metabolites Found in Dog Urine Solvent Systema
_____ Metabolites
4-Dimethylamino-3.5-xylyl methylcarbamate 4-Dimethvlamino-3,5-xylenol
2.6-Dimethylhydroquinone 2,6-Dimethyl-p-benzoquinone 4-Dimethylamino-3,5-xylyl
of ferrous hydroxide and then with Xvater.) T h e ether extract [ l ] contained 8.4Y0 of the total activity. T h e aqueous phase [2] contained 91.67,. Ether Extract [l]. T h e fractionation procedure for the ether-soluble material is shown in Figure 1. T h e ether [ l ] was dried over anhydrous magnesium sulfate and concentrated in vacuo a t heated and filtered hot. 'The filtrate \vas cooled but no crystals formed. The solution was concentrated, the glucuronide was regenerated with 5 ml. of concentrated ammonia? and the solution was evaporated to dryness. .4 large-scale paper chromatogram using solvent I \vas run on the residue [2C]. The activity was spread over the length of the paper. 'l'he chromatogram was cut at R, 0.10. 0.26. and 0.61. and each of the four fractions (2E. 2F. 2G. and 2 H ) \vias eluted from the paper Ivith methanol and concentrated. Paper chromatography of each fraction with solvent IX indicated the presence of four separate compounds: 12E] contained activity located at R,, 0.1 1 to 0.15, [ 2 F ] a t R, 0.09 to 0.13 and 0.45 to 0.49. [2G]a t R , 0.47 to 0.51 and 0.67 to 0.71. and [ 2 H ] a t H, 0.68 to 0.72 and 0.83 to 0.87. 'I'hese data show that thprr was overlapping in adjacent fractions. Fractions (2E. 2F. 2G. and 2 H ) were combined and hydrolyzed at 100' C. for 3 hours with 1 .5.V HCl. 0.01M in SnClr. Paper chromatography of 1211 \vith solvent I1 gave the follo\ving distribution
of activity: R, 0 to 0.02, (4-dimethylamino-3,5-sylenol), R, 0.06 tu 0.1 1 (probably represents unhydrolyzed glucuronides), R, 0.20 to 0.25 (2.6dimethylhydroquinone), R, 0.29 to 0.34 (unhydrolyzed 4-dimethylamino-3.5sylyl sulfate), and R, 0.85 to 0.89 (2.6dimethyl-p-benzoquinone), 'The hydrolyzed material \saa extracted Yvith peroxide-free ether and [ 2 J ] was chromatographed on paper ivith solvent 11. 'The activity coincided with 2,6dimethylhydroquinone and 2.6-dimethyl-p-benzoquinone. 'This quinone is an artifact as explained by \.Yilliams cf ai. 17). 'I'hr acid solution 12x1 was made alkaline with sodium bicarbonate and extracted with peroxide-free ether-. Paper chromatograms of [ 2 L ] ere ~ L L I I using solvents I , X . and XI. O n each paper the activity coincided with 4dimethylamino-3,s-xylenol. Identification of Sulfates. T h e fractionation procedure for thc watersoluble material 1201 is shown i n Fignrt. 4.
'l'he top phase [ 2 0 ] resulting from thr centrifugation during the glucuronidr isolation was made alkaline with ammonia and extracted three times with chloroform to remove the p-toluidine. 'I'he aqueous phase was concentrated in vacuo at
