IN SE C T I C I DE METABO L I SM
Nature of Toxic Metabolites Formed in Mammals, Insects, and Plants from 3(Dimethoxyphosphiny1oxy)-N,N-dimethylckrotonamide and Its N-Methyl Analog
R. E. MENZER' and J. E. CASIDA2
Department of Entomology, University of Wisconsin, Madison, Wis.
Bidrin, 3-(dimethoxyphosphinyloxy)-~,N-dimethyl-cis-crotonamide, i s metabolized to yield trace amounts of 3-(dimethoxyphosphinyloxy)-~-methyl-N-hydroxymethyl-cis-crotonamide and larger amounts of 3-(dimethoxyphosphinyloxy)-~-methyl-cis-crotonamide(SD 9 129). SD 9129 is further metabolized to yield 3-(dimethoxyphosphinyloxy)-N-hydroxymethyl-cis-crotonamide and 3-(dimethoxyphosphinyloxy)-cis-crotonamide. The toxicity to both insects and mammals increased upon successive N-demethylation. Balance studies on the fate of the P32 and C14 from Bidrit~-P~~, Bidrin-N-meth~1-C~~~ SD 9129-P32, and SD 91 29-N-rneth~1-C~~ are considered. Studies on milk residues, urinalysis, and metabolism in houseflies (Musca dornestica L.) and bean plants are reported. An unusual pattern of synergism of the toxicity of the Bidrin metabolites in houseflies by sesamex [2-(2-ethoxyethoxy)ethyl-3,4-(methylenedioxy)phenyl acetal of acetaldehyde] was noted.
T
wo vinyl phosphates which have recently been found very effective as agricultural insecticides are 3-(dimethoxyphosphinyloxy) - A'.-%' - dimethyl - cis - crotonamide (Bidrin, Shell Chemical Co. trade-mark) and 3(dimethoxyphosphinyloxy)- 1%' - methylcis-crotonamide (SD 9129. code designation. Shell Development Co.) Related vinyl phosphates already in use are mevinphos. M here the active ingredient is 3 - (dimethoxyphosphiny1oxy)methyl cis-crotonate. and phosphamidon, which consists of the mixed isomers of 3-(dimethoxyphosphinyloxy) - diethyl2-chlorocrotonamide Studies on the metabolism of mevinphos have established that hydrolysis can occur at the vinyl phosphate, P-0-methyl, or carbomethoxy group; these hydrolysis products are further degraded; and only the unhydrolvzed ester is highly toxic (9. 70, 23) Degradation of phosphamidon in plants involves the formation of the more toxic 3-(dimethoxyphosphiny1oxy)A' - ethyl - 2 - chlorocrotonamide (des.\'-ethyl phosphamidon), which must be considered \\hen evaluating residues of this pesticide Other degradation products identified include ,\..\--diethy1-2chloroacetoacetamide and .17-ethyl-2chloroscetoacetamide. The unsubstituted amide of phosphamidon [3(dimethoxyphosphinyloxy) - 2 - chlorocrotonamide] and the des-0-methyl derivative [3-(hydroxymethoxyphosphinyloxk) - .\..\ - diethyl - 2-chloro-
crotonamide] were not detected in plants (3, 76). Studies with 0,O-dimethyl 2,Z-dichlorovinyl phosphate or dichlorvos have established as metabolites in mammals the following compounds which lack the toxicity of the original molecule : inorganic phosphate, monomethyl phosphate, dimethyl phosphate, 0-methyl 2,2-dichlorovinyl phosphate, and dichloroethanol conjugates ( 7 7 , 75). The metabolism of Bidrin and SD 9129 was investigated in mammals, plants, and insects in relation to the mode of action and residue problems that might be anticipated. Only the ciscrotonamide isomers were considered, as these isomers are those of the greatest biological activity, and only small amounts. if any, of the trans-isomers are present in Bidrin or SD 9129. The nature of the phosphorus-containing hydrolysis products is not considered here, since a study on their nature following Bidrin administration to rats, cotton plants, and insects has been reported (5). The present investigation deals primarily with the fate of the dimethylamide or methylamide group on that portion of the administered dose that has not been hydrolyzed a t any given time after administration. It appeared possible that I\-demethylation might occur and that intermediates in the reactions leading to successive .V-demethylation \\odd be toxic (5: 78). Experimental
Present address, Department of Entomology, University of Maryland, College Park, Md. Present address, Division of Entomology and Acarology, University of California, Berkeley, Calif.
102
J. A G R . F O O D C H E M .
Materials. The following chemicals were provided by R. R. Whetstone, Shell Development Co., Modesto, Calif. : Bidrin, which was further purified by column chromatography; SD 9129. m.p. 49 ' C .; the unsiibstituted amide
analog of Bidrin and SD 9129, or 3 - (dimethoxyphosphinyloxy) - ciscrotonamide, m.p. 101' C . ; Bidrin acid, or 3 (dimethoxyphosphinyloxy) - G ~ S crotonic acid, prepared by hydrolysis of the cis-crotonate isomer of mevinphos according to the method of Spencer et al. (26) ; des-0-methyl Bidrin. or 3-(hydroxymethoxyphosphinyloxy) iV!.V dimethyl - cis - crotonamide, sodium salt, m.p. 141' C.? prepared by reaction of cis-Bidrin with sodium iodide according to Spencer et al. (26); A\\,AY-dimethylacetoacetamide, b.p. 83-85' C.jO.2 mm.; AV-methylace toace tamide, m. p. 44 '' C . ; 3 - hydroxy dimethylbutyramide, b.p. 88-89" C./0.75 m m . ; and 3hvdroxv-dV-methvlbutvramide, b.D. 110, , l i ' C . ~ O . mm. I Radiolabeled Bidrin and SD 9129 samples were also provided by the Shell Development Co. at the follo\ring specific activities: Bidrin-P3* a t 2.6, 2.8, 3.0. and 4.6 mc. per mmole; Bidrin-.Vmethyl-C'* at 1.0 mc. per mmole; Bidrin-O-methyl-C14 at 0.6 mc. per mmole; SD 9129-P3* at 2.1 mc. per mmole; SD 9129-.\7-methyl-C'4 at 2.0 mc. per mmole; and SD 9129-0methyl-CI4 a t 0.2 mc. per mmole. All radiolabeled compounds were purified on either silica gel or Celite partitioning columns. Only the cis-crotonamide isomers of the labeled materials \\ere iised after ascertaining their composition and purity by infrared spectra and cochromatography with authentic nonlabeled materials. Attempted Synthesis of .\-Hydroxymethyl Analogs of Bidrin and SD 9129. SD 9129 (3.2 mmoles). formaldehyde (25.1 mmoles in 2.0 ml. of water), and enough sodium bicarbonate to raise the p H to 7.0 were held 20 hours at 4.5" to 50' C., and the reaction mixture was then extracted with chloroform. The chloroform was evaporated and the residue chromatographed on the Cdite column as descrihed later.
-
-
-
-
,
I
Two products in addition to the starting material were eluted, one before the position of Bidrin and the second in the position indicated as unknown 4 (Figure 2). T h e t\vo products other than SD 9129 yielded formaldehyde as detected with a sulfuric acid solution of chromotropic acid ( 8 ) and inorganic phosphate follo\ving digestion with perchloric acid according to the method of .411en (2). The formaldehyde-yielding functional groups \\'ere present in organophosphorus compounds \vith both new products, since the phosphate- and formaldehyde-yielding materials cochromatographed in both cases. The material from the first peak contained no bands associated with hydroxyl functions in its infrared spectrum, while the material chromatographing in the unknown A region yielded the spectrum indicated in Figure 1. T h e yield of unknown A was 2OYG. This inaterial is proposed to be 3-(dimethoxyphosphiny1oxy)methyl - r\-. hydroxymethyl- cis-crotonamide, on the basis of the synthetic route, infrared spectrum. chromatographic characteristics, and degradation to yield formaldehyde and phosphate from thc same compound. An NMR spectrum \vas also consistent with the proposed structure. T h e unsubstituted amide analog of Bidrin (1.34 mmoles), formaldehyde (25.1 mmoles in 2.0 ml. of Ivattr), and enough sodium bicarbonate to raise the pH to 7.0 were held 20 hours at 28' C.. and the reaction mixture was then extracted with chloroform. T h e extract \vas purified in a manner similar to that for unkno\vn A to yield the unreacted unsubstituted amide and only one additional product, {vhich chromatographed in the position indicated for unknown B in Figure 2. This product, as u i t h unknown A. contained groups \vhich yielded both phosphate and formaldehyde upon degradation. The yield of unknoum B \vas 847,. This material is proposed to be 3-(dimethoxyphosphiny1ox)-) - *Y - hydroxymethylcis-crotonamide bised on the same crittria applied to unkno7vn A . These routes of synthesis were suggested in part by \Vhetstone (.30). The infrared and S h f R spectra were prepared and interpreted a t the Shell Development Co., Modesto, Calif., by G. E. Pollard. Based on rechromatography of the samples. the mono-X'-hydroxymethylamide appeared to be stable u p to 2 iveeks a t 4 ' C., but the .Y-methyl-*Yhydroxymethylamide was almost entirely decomposed after 4 weeks to yield SD 9129. T h e sample of LYmeth:-l - -Y - hydroxymerhylamide had partially decomposed to SD 9129 during shipment prior to preparation of the infrared spectrum of Figure 1. I n view of the kno\\-n ability of hydroxymethyl compounds to decompose Lvith loss of formaldehydt ( 2 9 ) :this is not surprising. A\--
\ I ,
loci
\I\ I \ I
q
2
Figure 1.
3
4
5
Infrared spectra
6
L J
7 8 9 1 0 1 1 WAELENcTn IN MKRONS
1
2
1
4
3
1
on cis-(CH30),P(0)OC(CH,)=CHC(O)R
4
1
5
1
I
6
compounds
Spectra determined from 10% solutions in methylene chloride using Beckman Model IR4 infrared spectrophotometer. Compounds were o f known structure except for unknowns A and B, synthetic materials o f the proposed structures. Unknown A contained approximately 50% SD 9 1 29; unknown B contained little, i f any, o f the unsubstituted amide
Partitioning Characteristics of Bidrin and Potential Metabolites. Partition coefficients for the phosphorus-containing compounds between chloroform and water were determined by either total phosphate or radioactivity analysis. Other partition coefficients were estimated on the basis of the weight of materials recovered upon extraction with chloroform. The following partition coefficients (chloroform/\vater) a t 25' C. \vere obtained: Bidrin. 25; SD 9129, VOL.
13
2.5; the ,V-methyl-.\~-hydroxymethylamide analog of Bidrin. 0.97; the unsubstituted amide: 0.40; the .V-hydroxymethylamide analog of SD 9129, 0.12; A\~,i\7-dimethylacetoacetamide. 1.15; .Ymethylacetoacetamide: 0.17 ; 3-hydroxy.V,,V-dimethylburyramide. 0.80; and 3hydroxy-.Y-methylbutyramide. 0.017. Chloroform was used to extract metabolites from aqueous solution for analysis of residues in plants and urine as described later. Three extractions NO. 2 M A R . - A P R .
1965
103
with chloroform volumes equal to the aqueous volume were used. Recoveries anticipated from this extraction procedure if the partitioning were unaffected by the biological material would be 99.99% for Bidrin. 97.7% for SD 9129. 86.9% for the .Y-methyl-,V-hydroxymethylamide analog of Bidrin, 63 6% for the unsubstituted amide, 28.87' for the ,V-hydroxymethylamide analog of SD 9 12 9, 89.9% for .V,-V-dimethylace toacetamide, 37.5% for ,V-methylacetoacetamide, 82.8% for 3-hydroxy-1V,.Vdimethylbutyramide, and 4.97' for 3hydroxy-.\-methylbutyramide, Neither Bidrin nor SD 9129 was extracted to any extent into n-hexane from water. These partitioning characteristics suggested the use of a partitioning column and mixtures of chloroform and hexane for resolving these potential Bidrin metabolites. Phosphorus-containing hydrolysis products of the Bidrin analogs would be expected to remain in neutral aqueous solution on extraction with chloroform. Based on such a partitioning procedure and total phosphorus analyses, the following half-life values were obtained for the organophosphates at 0.170 in 0 0 3 7 s sodium hydroxide a t 28" C.: 64 minutes for Bidrin, 42 for SD 9129. and 35 for the unsubstituted amide. Chromatography and Detection of Metabolites, Celite or silica gel was used in 2- X 30-cm columns for separation of metabolites or 3.5- X 30-cm. columns for larger scale purifications. For the standard 2- X 30-cm. columns, water was added to silicic acid (Mallinckrodt chromatographic grade, 100mesh) in a ratio of 1 to 2 (v. :w.)and mixed until the silica gel was homogeneous. For the other type of column, 25 ml. of water were added to 40 grams of Celite (Johns Manville's Hyflo SuperCel) a r d mixed until the damp powder \\as homogeneous. The prepared stationary phase was then slurried in n-hexane and used in the packing of the columns. Silica gel was used in the early stages of this study, but in changing to different lots of silicic acid variable decomposition of metabolites and poorer resolution resulted. In most of the studies reported and for all quantitative data tabulated, CeIite columns were utilized. Compounds were eluted from both silica gel and Celite columns with hexane, followed by hexane-chloroform mixtures, then chloroform, and finally a wash of methanol to clear the columns of any material that might have remained after elution with the hexane and chloroform mixtures. Resolution on the Celite columns is indicated in Figure 2. The ratio of eluting solvents was slightly different for the silica gel columns, although the basic relationships and order of compound elution remained the same. Twenty-milliliter fractions were collected a t a flow rate of approximately 2 ml. per minute. I n some cases P32- and i\r-methyl-C14labeled compounds were mixed for treatment of the organisms. The energy
104
J. A G R . F O O D C H E M .
Figure 2. Chromatographic characteristics of some potential Bidrin metabolites on a Celite column based on partitioning between water and hexane-chloroform mixtures Mixture of 50 mg. o f each phosphorus-containing compound was chromatog r a p h e d and 0.2 ml. from each 20-ml. fraction was analyzed for total phosphorus content (2). 50 mg. of each non-phosphorus-containing compound w e r e similarly chromatographed a n d 1.0 ml. from each 20-1711. fraction was analyzed b y a colorimetric procedure based on the 2,4dinitrophenylhydrazones o f the acetoacetamides, while the elution positions o f the 3-hydroxybutyramides were determined b y weights on the residues following evaporation of the solvents. All compounds were o f known structure a n d high purity except unknowns A and 8, which were synthetic materials
difference in the beta particles of P32 and CI4 made it possible to assay \vith a liquid scintillation spectrometer each of these isotopes separately with little interference from the other in a mixed label sample. In this manner metabolism of both the phosphate and the .V-methylamide portions of the molecule could be investigated simultaneously under identical biological conditions. Known nonradioactive compounds suspected of being metabolites were added to columns along with radiolabeled samples from treated plants or animals to aid in establishing the identity of the radiolabeled compounds with known maTerialS eluting from the columns. Colorimetric procedures were used for total phosphate (2). formaldehyde (8),and 2,4 - dinitrophenylhydrazones derived from the known compounds. For determination of the ,Y,.V-dimethylacetoacetamide and 'V-methylacetoacetamide which might result from hydrolysis of Bidrin and SD 9129, respectively, a rapid qualitative test was used, followed by quantitative analysis on the appropriate regions of the column eluates. T o locate the regions where these two acetoacetamides were eluted, a 0.5-ml. aliquot of each chloroform or hexane-chloroform fraction 1% as transferred to a small test tube and 2 drops of a 1.0% solution of ferric chloride in chloroform were added. Upon the addition of 1 drop of pyridine, a purple color was developed if either of the acetoacetamides was present (24). The
acetoacetamides \vere then quantitatively determined as colored derivatives formed by degradation of the 2.4-dinitrophenylhydrazones (75). The 2,4-dinitrophenylhydrazones were prepared by adding 1.0 ml. of a 0.1% solution of 2!4dinitrophenylhydrazine in 2,V hydrochloric acid to the residue from evaporation of 1.0-ml. aliquots from column fractions, followed by heating a t 90' C. for 30 minutes. The addition of 7.0 ml. of 0.75N sodium hydroxide formed a reddish-purple material which tended to precipitate. An equal volume of 2methoxyethanol dissolved the precipitate. The absorbance of the resulting solution \vas read at 443 mp. The chromatographic positions of the reduction products of the two acetoacetamides, 3-hydroxy-S.S-dimethylbutyramide and 3-hydroxy-iY-methylbutyramide. were determined with ceric ammonium nitrate reagent which was changed from yellow to deep red in their presence (24), or by evaporation of the fractions and determination of weights and infrared spectra of the residues. Formation of Radiolabeled Derivatives from Degradation Products of Metabolites. To determine the nature of the amide portion of certain CI4metabolites of Bidrin and SD 9129. the compounds under investigation were subjected to hydrolysis, derivative formation, and reversed isotope dilution procedures. Metabolites of Bidrin-.Vmethyl-CI4 were used for determination of radio-labeled methylamine and form-
aldehyde, or dimethylamine; metabolites of SD 9129-.V-methyl-CI4, for determination of radio-labeled formaldehyde or methylamine. Following chromatography, the solvent was evaporated from a known quantity of resolved metabolite contained in a roundbottomed flask. For the determination of labeled methylamine or dimethylamine. 10 ml. of 2Al' sodium hydroxide were added to the flask. which was then fitted with a water-cooled distillation condenser leading to a cooled receiving tube. The sodium hydroxide solution of the metabolite was heated until nearly all of the water containing hydrolyzed C14 compounds had distilled. T o the distillate were added 20 pl. of a 40Yc aqueous solution of methylamine. folloued by 42 pl. of phenyl isothiocyanate. This mixture was vigorously shaken. Methyl phenylthiourea crystallized (m.p. 113' C., 70% yield) from solution when held 18 hours a t 4' C. T h e C14 content of a portion of the crystals was determined, and the remaining thiourea was subjected to thin la Ver chromatography on silica gel plates (Silica Gel G, Brinkmann Instruments, Inc., Great Neck. N. Y . ) using 2 to 1 etherhexane mixture. Metlivl phenylthiourea. R, 0.5, and dimethyl phenyl thiourea, R, 0.2 in a separ'ite determination. were detected by spraying with 1.O% aqueous ferric chloride followed by 1.O% aqueous potassium ferricyanide to yield blue spots (72). The coincidence of the radioactive material with methyl phenylthiourea was determined by radioautography. A procedure was developed for determination of labeled formaldehyde, methylamine, and dimethylamine recovered on acid hydrolysis of a single sample of S-methyl-Ci4-metaboli te. For C14-formaldehyde determination the initial procedure was the same as above, except that 2.0% hydrochloric acid was used instead of 2-V sodium hydroxide. T o the distillate were added 5 pl. of a nonradioactive 37% aqueaus solution of formaldehyde and 25 ml. (of a hot solution of 0.25y0 aqueous 4-hydroxycoumarin. and the mixture was refluxed for 1 hour. The white crystals of dicoumarol (m p. 285' C:, yield 68%) were removed by filtration of the hot solution (22), and the radioactivity was determined. Unlabeled methylamine and dimethylamine (0.10 mmole each) were then added to the filirate irom the dicoumarol preparation, and the solution was evaporated to about 1 ml. with a stream of air and an infrared lamp. A sufficient quantity of 107-l-C’4-phenylthiourea but no dimethyl-C14-phenylthiourea. Unknown .4 cochromatographed with the S-methyl.V-hydroxymethylamide analog of Bidrin formed by reaction of SD 9129 Jvith formaldehyde. Unknown A is therefore proposed to be 3-(dimethoxyphosphinyloxy) - -1’- methyl - .V - hydroxymethyl-cis-crotonamide. Cnknown B \vas formed from both Bidrin and SD 9129 and contained one half the C14:P32 ratio administered \\.hen derived from
Table 1.
Metabolites of B i d r i t ~ - Pand ~ ~ SD 9129-P32in Urine Following Treatment of Mammals at 1.0 Mg. per Kg. (All results expressed as per cent of administered P32)
Treatment and Hours
3 -
-n-c,-
0 0-6 3 6-12 0 6-12 3 & Q 12-24 2 8: 0 24-48 3 0-48 Q 0-48
NO.of Animals
I,
5 6 5 11 11 2 2
Total Excreted
Hydrolysis Productsa
5 1. -
4 44 8 12 5 11 0 0.40
2.1 70.1 64 5
37 9
cis-(CH30)zP(O)OC(CHa)=CHC(O)R N(CH3)z
N(CHz0H)CHa
NHCH3
Rats, Bidrin (Oral by Stomach Tube) 3 0 1 3 7 9
13 8 10 8 10 3 0.38 2.05 60.5 53.1
3 0 0 39 0 31 0.003 0.02 2.8
3.2
0 40 0 25 0 08 0,002 0,0006 2.8 0.08
5 4 0 84 0 16 0.01 0.03 3.0 5.3
NHCH20H
0 68 0 23 0 07 0 01 0 001 0.00 0.35 0.09
NHz
0 00 0 00 0.00 0 00 0 00 0.00 0 00 0.00
CH,OH FractionC
0 67 2 0 0 12 0 15 0 003 0,003 0.68
2.7
Rats. SD 9129 (Oral by Stomach Tube) 3 0-6 0 0-6 3 6-12 0 6-12 3 8: 0 12-24 3 & 9 24-48 3 0-48 9 0-48
6 6 6 6 12 12 2 2
53.6 57.8 13.4 5.1 3.0 71.3 62.8
34.5 34.6 7.5 11 . I 4.4 2.5 52.2 48.2
0-48
19
71 . 7
57.0
9.0
...
...
11.6 17.3 0.81 1.3 0.47 0.37 13.0 12.1
1.8 1.6 0.07 0.17 0 07 0.04 1.6 0.64
Mice, Female, Bidrin (Intraperitoneal) 1.2 0.43 9.9
1 .o
0.00
2 2
0.04
0.04
0 36
0.31
0.00
0.77
0.00 0.16
0.003 0.53
0.01 0.48
... ... ,
.
I
... ...
...
...
... ...
... ...
...
... *..
0 24 0.06 0.03 0.03 0.00 0.00
0.20 0.27
5.5 4 2 0.6 0.8 0.2 0.1 4.3 1.6
Rabbits, Male, Bidrin (Intraperitoneal) 0-48
2
72.1
0-48
2
97.6
0-1 1-2
1 1
0.05 10.6
67.7
1 .o
0.18
2.8
Dogs, Female, Bidrin (Oral by Stomach Tube) 92.2
0.76
0.28
3.3
Goat, Female, Bidrin (Oral by Capsu1e)d 0.023 7.8
0.0O06 0.10
0.014 1.5
0.00 0.00
a Hydrolysis products considered to be P32compounds remaining in water on extraction with an equal volume of chloroform in Bidriii studies. or three extractions with equal volumes of chloroform in SD 9129 studies. b Bidrin is the --N(CH,)? analog and SD 9129, the -NHCHa analog. Percentages based on column chromatography of chloroform extracts as indicated in a. Elesults corrected for incomplete extractions of certain metabolites based on their partitioning characteristics as indicated in experimental section. c Methanol fraction eluted from columns contained a mixture of unidentified materials. d Urine from Bidrin-treated goat collected at intervals greater than those indicated contained all P3*compounds as hydrolysis products. compounds. A11 urine samples from SD 9129-treated goat contained no chloroform-extractable
Bidrin and the same C14:P32 ratio administered \\.hen derived from SD 9129. Acid hydrolysis yielded 1 mole of formaldehyde-C14 per mole of compound. but no labeled amine. L-nknown B cochromatographed with the .V-hydroxymethylamide analog of SI)'9129 formed by reaction of the unsubstituted amide with formaldehyde. Unknown B is therefore proposed to be :3-(dimethoxy- hydroxymethylphosphinyloxy) - .Icis-crotonamide. T h e final peak eluting \vith methanol, present in extracts of organisms treated xvith either Bidrin or SD 9129. yielded variable C14:P32 ratios. depending on the biological system. It therefore was not a homogeneous fraction. Known compounds eluting in this position are Bidrin acid, des-0-methyl Bidrin, and 3hydroxy - .V - methylbutyramide. The methanol column \\.ash may contain some of these compounds. and perhaps others of a hydrolytic nature, but no attempt was made to characterize these matrri,rls further. Chemical Nature of Bidrin- and SD 9129-.lT-methyl-C14 Metabolites. Hydrolysis of Bidrin-.V-methyl-~ll qhould
yield .V..V-dimethyl-Cl4--acetoacetamide. and of SD 9129-.V-methyl-C14 should yield :Y-methyl-C14-acetoacetamide. In vivo reduction might subsequently dimethyl - C14 form 3 - hydroxy - ,?'.itTbutyramide and 3-hydroxy-.Y-methylC14-butyramide. The chromatographic resolution was adequate to investigate the potential presence of the two acetoacetamides as components separated from phosphorus-containing materials. but \\Vas less satisfactory for the 3hydroxybutyramides since the .Y,-Ydimethyl analog eluted in the approximate position of SD 9129 and the >Irmethyl analog eluted in the methanol fraction (Figure 2). Studies with mixtures of labeled materials were particularly useful with 3-hydroxy-.V..17-dimethylbutyramide since a higher than anticipated C14:P32ratio for this peak indicated the presence of the butyramide as a contaminant. The methanol fraction might consist of a mixture of phosphoriis- and non-phosphorus-containing metabolites. The heterogeneity of metabolities in the methanol fraction \vas confirmed with mixed label studies \.\.here the C14:P32 ratio recovered varied VOL.
from that administered as follo\vs: almost the same for urine from SD 9129treated rats; 1.5 times for urine from Bidrin-treated rats; 0.4 times for urine from a Bidrin-treated goat; 0.5 times for urine from Bidrin-treated mice ; and 1.8 to 16.3 times for milk from a goat treated with Bidrin or SD 9129, as diScussed later. In early samples of urine from rats treated lvith Bidrin-,Y-methyl-C14. evidence \vas obtained of the presznce of .V - methyl - CI4 - acetoacetamide and 3 - hydroxy - -V..V - dimethyl - CI4butyramide. There \vas no indication of .lr3sV - dimethyl - CI4 - acetoacetamide in urine of Bidrin-treated rats. nor of .V-methyl-C14-acetoacetamide in urine of SD 9129-treated rats. None of these materials appeared in other urine samples or in the milk samples folloiving either Bidrin- or SD 9129-.V-methylC14 administration. In the 0- to 8-hour sample of iirine from male rats treated \vith Bidrin-.V-methyl-C14.a shoulder on the last-elutiqg portion of the SD 9129 peak was suspected of being 3-hydroxyAY..V-dimethyl-C14-butyramide. This labeled compound cochromatographed
1 3 , NO. 2 M A R . - A P R .
1965
107
Table It.
Bidrin, SD 9129, and Organoextractable Metabolites in Milk of a Goat Following Oral Administration of B i d r i ~ ~ or - P SD ~ ~ 9129-P32in Capsules at 1.0 Mg. per Kg. Tofal
P.P.M. Hr. offer Treafmenf
Ports per Million Based on Column Chromafographya NHCH3' NHCHzOH
~
N(CHjI!"
NHz
CHaOH frac.
,0001 0.0011 0.0028 0.0013
