LARSEN, BAKKE LITERATURE CITED Burger, K. M., Fresenius' 2. Anal. Chem. 224,425 (1967). Davison, K.L., U S . Department of Agriculture, Agricultural Research Service, Metabolism and Radiation Research Laboratory, Fargo, N.D ., personal communication, 1974. Davison, K. L., Sell, J. L., Arch. Enuiron. Contam. Toxicol. 2,
Inoi, T., Gericke, P., Horton, W. J., J. Org. Chem. 27, 4597
Feil, V. J., Lamoureux, C. H., Styrvoky, E., Zaylskie, R. G., Thacker, E . J., Holman, G . M., J . Agric. Food Chem. 21, 1072
Received for review September 6, 1974. Accepted January 17, 1975. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the US. Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.
(1962).
Loev, B., Goodman, M. M., Chem. Znd. (London),2026 (1967). Paulson, G. D., Poultry S c i . 48,1331 (1969). Sell, J. L., Davison, K. L., Fed. Proc., Fed. Amer. Soc. Enp. Biol.
32,2003(1973).
222 (1974). (1973).
Haegele, M . A,, Tucker, R. T., Bull. Enuiron. Contam. Toxicol.
11,98 (1974).
Metabolism of 2-Chloro-4-cyclopropylamino-6-isopropylamino-striazine (Cyprazine) in the Rat Gerald L. Lanen* and Jerome E. Bakke
Rats given single oral doses of ringJ4C-labeled cyprazine (I) excreted 97.6% of the dose within 72 hr (urine, 72.7%; feces, 24.9%). Less than 0.1% of the radioactivity was detected as l4CO2 in the expired air. The rat carcasses contained 7.5% of the radioactivity a t sacrifice (72 hr after dosing). Four urinary metabolites were identified by comparison of their mass spectra with those of authentic compounds. These compounds and the percentages of the urinary radioactivity that they represented were: 2-hydroxy-4,6-diamino-striazine (11, 3.0%); 2-chloro-4,6-diamino-s(111, 13.9%); 2-hydroxy-4-aminotriazine
6-isopropylamino-s-triazine (IV, 5.3%); and 2-chloro-4-amino-6-isopropylamino-s-triazine (VII, 2.1%). Two fecal metabolites were identified as 11 and IV. Four additional urinary metabolites were characterized by mass spectrometry as 2-chloro-4-amino-6-[(2-hydroxy-1-methylethyl)amino]-s-triazine (V, 5.9%); 2-chloro-4-amino6-cyclopropylamino-s-triazine (VI, 7.7%); [ 2 -chloro -4-cyclopropylamino-s-triazinyl( 6)lalanine (VIII, 9.9%); and 2-chloro-4-cyclopropylamino-6 - [ (2-hydroxy-l-methylethyl)amino]-s-triazine (IX, 1.6%).
Bohme and Barr (1967) identified rat and rabbit urihydrolysis of the 2-chloro group that occurs with the use nary metabolites from the 2-chloro-s-triazines: simazine of ion-exchange chromatography. [2-chloro-4,6-bis(ethylamino)-s-triazine]; atrazine (2-chloEXPERIMENTAL SECTION ro-4-ethylamino-6-isopropylamino-s-triazine);and propazThe meine [2-chloro-4,6-bis(isopropylamino)-s-triazine]. Chemicals. 2-Chloro-4-cyclopropylamino-6-isopropyltabolites of these 2-chloro-s-triazines were all 2-chloro anamino-s-triazine-W, uniformly labeled in the ring (9.4 alogs of their respective parent molecules. Mono- and pCi/mg), and unlabeled cyprazine were provided by Gulf di-N-dealkylation and oxidation of the N-alkyl groups to Research and Development Co., Merriam, Kan. Radiopuacids were major routes of metabolism for these 2-chlority of the radioactive chemical was greater than 99.0% as ro-s-triazines. determined by thin-layer chromatography (TLC). AuBakke et al. (1972) identified four and characterized thentic standard compounds were obtained from Geigy two metabolites from atrazine in rat urine. As isolated, Chemical Corporation. these urinary metabolites were identified as 2-hydroxy anAnimal Treatment. All rats were dosed by stomach alogs of atrazine. However, these metabolites were isolattube with r i ~ ~ g - ~ ~ c - l a bcyprazine eled dissolved in 0.5 ml of ed by ion-exchange chromatography at elevated temperaethanol. Urine and feces were collected at 24-hr intervals. tures, and subsequent investigations indicated that these Two rats (each weighing 310-360 g) were each given 0.5 metabolites may have been artifacts. For instance, when mg of cyprazine containing 0.93 fiCi of I4C as a single oral atrazine and 2-chloro-4,6-diamino-s-triazine were placed dose. These rats were housed in all-glass metabolism on the ion-exchange column used in their isolation procecages, and COz was collected as previously reported (Bakke dure, both compounds were quantitatively recovered from et al., 1967). After 3 days, the rats were sacrificed, and total the ion-exchange column as their 2-hydroxy analogs. carcass radioactivity was determined. Bakke et al. (1972) concluded that any 2-chloro-s-triazine Twelve rats (each weighing 300-420 g) were each given metabolites present in the urine were quantitatively con5.0 mg of cyprazine containing 0.51 gCi of I4C as a single verted to 2-hydroxy analogs. However, the presence of oral dose. These rats were housed in stainless steel cages, 2-hydroxy-s- triazine metabolites in the urine could not be and urine was collected daily and pooled. Feces were also ruled out. collected daily and pooled. The pooled urine and pooled This report describes the metabolism of the 2-chloro-sfecal samples were assayed for radioactivity and used for triazine cyprazine (2-chloro-4-cyclopropylamino-6-isopro- metabolite isolation. After 3 days, the rats were sacrificed. pylamino-s-triazine, I) by the rat. The urinary metabolites Instrumentation a n d Quantitation of Radioactivity. were isolated by using paper chromatography to minimize All column eluates were monitored with a Packard Model 320E liquid scintillation flow system. With aqueous eluates, anthracene was used as the scintillator in the flow cell, and with organic eluates, cerium-activated lithium United States Department of Agriculture, Agricultural glass beads were used as the scintillator in the flow cell. Research Service, Metabolism and Radiation Research Radioactive components on paper chromatograms and Laboratory, Fargo, North Dakota 58102. 388
J. Agric. Food Chem., Vol. 23,No. 3, 1975
METABOLISM OF CYPRAZINE RAT URINE (0-24hr. wmplr)
I
Figure 1 .
thin-layer chromatograms were located with a Packard Model 7200 chromatogram scanner. Liquid scintillation in counting solution A (Bakke et al., 1967) was used to quantitate radioactivity with a Nuclear-Chicago Mark I spectrometer. Combustion of freeze-dried samples (100-200 mg) was used to quantitate carcass and fecal I4C, with a Model 305 Packard Tri-Carb sample oxidizer. Mass spectra were obtained with a Varian M-66 mass spectrometer equipped with a V-5500 control console; a solid-sample inlet system was used. Chromatographic Methods. Amberlite XAD-2 (Mallinckrodt Chemical Works) was equilibrated with water in a 1.5 x 25 cm column. The XAD-2 column was conditioned by a two-column volume wash with methanol followed by a two-column volume wash with water. This process was repeated three times. With the column equilibrated with water, the samples (10 ml) of rat urine were applied to the column under nitrogen pressure. The column was first washed with three-column volumes of water, and the radioactivity was then eluted with methanol. The cation-exchange column (AG 50-X8 [H+]) was converted to the ammonia form with 1 N N H 4 0 H and washed free of base with water. Acidified (pH 2.0) aqueous samples were applied to the column, and the column was washed with three-column volumes of water. The radioactivity was then eluted from the column with 1 N NH40H, The anion-exchange column (AG 1-X8 [OH-]) was washed with water before the samples were applied. Aqueous samples were applied under nitrogen pressure, and the column was washed with three-column volumes of water. The radioactivity was then eluted from the column with 1NNH4HC03. The paper chromatograms were all developed with the organic phase from the mixture of isoamyl alcohol-concentrated NH40H-H20 (40:10:50, v/v/v); Whatman No. 1 paper was used. Thin-layer chromatography was accomplished with prc-
coated silica gel thin-layer plates (0.5 mm thickness, Brinkman No. 5769/0020). Three solvent systems were used: solvent 1, CHCl3-ethanol (5:1, v/v); solvent 2, l-butanol-H20-concentrated NH4OH (4:5:1, v/v/v); and solvent 3, methanol-concentrated NH40H (9:1,v/v). Gas-liquid chromatography (GLC) was performed with a 6 ft x 1h in. i.d. glass column packed with 3% SE-30 on 60-80 mesh Chromosorb W in a Perkin-Elmer 801 gas chromatograph fitted with an effluent splitter (10% to the flame ionization detector). The oven temperature was programmed at either lO"/min or 6"/min from 100 to 250"; the injector temperature was maintained at 200". The detector was located within the oven. The carrier gas was helium at a flow rate of 30 ml/min. Metabolite Isolation. The flow diagram shown in Figure 1 lists the combinations of methods used to isolate each metabolite from rat urine. Aliquots of low specific activity (5.0-mg doses, 0- to 24-hr rat urine) were placed on the XAD-2 column. The radioactivity that eluted from the XAD-2 column with methanol was concentrated and spotted in a continuous band on paper. The developed paper chromatograms were cut into seven radioactive sections (A-G, Figure l), and each section was extracted with methanol. The radioactivity extracted from each section was further purified by using the TLC solvent systems specified in Figure 1. The radioactive areas were scraped from the TLC plates and the radioactivity was extracted from the silica gel with methanol. These extracts were taken to dryness and derivatized by heating to 80" for 16 hr with an excess of N,N-bis(trimethylsily1)trifluoroacetamide containing 1% trimethylsilane (Regisil, Regis Chemical Co.) . The silanized metabolites were gas chromatographed and the elution of radioactivity was monitored by trapping each peak in a glass tube and assaying the radioactivity washed from each tube. Feces were extracted sequentially with hexane, ethyl acetate, acetone, methanol, and water in a soxhlet apparatus, and the extracted radioactivity was determined. J. Agric. Food Chem., Vol. 23,No. 3, 1975
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LARSEN, BAKKE
The methanol extract was taken to dryness, dissolved in 5 ml of water, acidified to pH 2, and applied to a cation-exchange column. The radioactivity eluted with 1 N N H 4 0 H was freeze-dried, dissolved in 5 ml of water, and applied to the anion-exchange column. The radioactivity was eluted from the anion-exchange column with 1 N NH4HC03 and freeze-dried. The bicarbonate salts were removed from the radioactivity by dissolving the residue in water, acidifying to pH 2, and chromatographing the radioactivity on the cation-exchange column. The bicarbonate salts were eluted from the column with three-column volumes of water. The radioactivity was eluted with 1 N NH40H, freeze-dried, and spotted on a paper chromatogram. The radioactive areas on the developed paper chromatogram were extracted from the paper with methanol, and the methanol extracts were taken to dryness, derivatized with Regisil, and gas chromatographed 1
Table I. Recovery of Administered Radioactivity from Rats after a Single 0.5-mg Dose of Ring J4C-Labeled Cyprazine
% of "C dose Hour
Urine
24
61.4 4.3 7 .O 72.7
48 72
Total
Feces Carcass Hide 21.0 2.3 1.6 24.9
6.4
6.4
1.1 1.1
CO,
