Khere the range is given, the compound under test was run at various concentrations and Beer's law was found to apply fairly m-ell. EXTRACTION OF MEPROBAMATE F R O M URINE
Any nonspecific method such as the one described here has the disadvantage that the compound to be determined must be separated from others of a similar nature before the method can be applied. This was accomplished for meprobamate in urine as follows. Extract the meprobamate from 4 ml. of urine by shaking vigorously with 30 ml. of ether for 90 seconds in a 125-ml. separation funnel. Allow the layers to separate for 2 to 3 minutes and drain off the aqueous phase completely along with a little of the ether. K a s h the ether layer twice with 3 to 4-ml. portions of water, shaking for a few seconds during the second ryash. After allowing ample time for complete separation of the water phase, drain this off along with a little ether and pipet out a 15-ml. aliquot of the ether and run it through a column of granular anhydrous potassium carbonate. The column is 7 mm. in diameter and 25 to 30 cm. in length and is supported by a plug of glass ~ o o at l the bottom. It is convenient to use a tube n idened a t the top so that 15 ml. can be run a t once. The column is filled dry and is not packed. so gravity flow is sufficient. As soon as the ether meniscus reaches the top of the column, add 10 ml. of ether to nash the sample through.
Collect all of the ether in a 50-ml. Erlenmeyer flask. Evaporate the ether on a steam bath and dissolve the residue in 5 or 10 ml. of water depending on the expected concentration of meprobamate. Use a 1-ml. aliquot for the determination. The recovery of meprobamate added to urine was 87% for one lot of Mallinckrodt potassium carbonate and the loss on the column was negligible. For another lot of potassium carbonate the recovery was 82%. A recovery should be run and the resultant correction factor should be applied to the results. The blank values for urine from healthy human males (16 individuals) ranged between 0.5 and 7.7 expressed as micrograms of meprobamate per milliliter of urine. The method should readily be applicable to gastric contents since r a t gastric juice gave a blank of zero. The reproducibility of the method was tested by determining the recovery of 80 y meprobamate per ml. of urine on 12 successive samples, running the colorimetric determination in duplicate. The percentage recovery ranged between 80.i and 84 with a mean of 82.1. The variability expressed as standard deviation was =t1.05, using SD = i(dev. from mean)2
1
n - 1
Kalkenstein and his coworkers, using a colorimetric procedure for meprobamate based on a difference principle (4,
Table 1. Suitability of the Method for Use with Various Compounds
%
Compound n,L-Alanine Lysine, HC1 p-Aminobenzoic acid Sulfanilamide Acetanilide Urea Urethane Meprobamate Guanidine. H2C03 Uric acid Creatine. H20
of Complete- Range, ness Y 33 110 77 112 107 59 50 38 67 87 44
2-20 2-10 2-20 2-20 2-20 1-10 2-20 5-40
S o t tried Piot tried Nottried
found 23 samples of human urine, taken from subjects on a n excretion study, to have a n average concentration of 38.5 y per ml. Using the present method and the same samples, the authors found a n average concentration of 36.8 y per ml. LITERATURE CITED
( I ) Pan, S. C., Dutcher, J. D., ~ZSAL. CHEU.28, 836 (1956). (2) Pryde, D. R., Soper, F. G., J . C h e w SOC.1931, 1510. (3) Rydon, H. N., Smith, P. W. G., A'ature 169, 922 (1952). (4) Walkenstein, S. S., Knebel, C. Pi,, MacMullen, J. A., Seifter, J., J . Pharmacol. Exptl. Therap. 123, 254 (1958). RECEIVED June 17, 1958. Accepted December 29, 1958. 2nd Regional Meeting, ACS, Delaware Valley, Philadelphia, Pa., February 5, 1958.
PoIa rogra phic Reduc ti o n of Nonconiugated Steroida I Ketones PETER KABASAKALIAN and JAMES McGLOTTEN Chemical Research and Development Division, Schering Corp., Bloomfield, N. J.
b The direct polarographic reduction of nonconjugated ketones in various positions on both the nucleus and the side chain of a steroid molecule has been carried out. The polarographic reduction of hydroxyl and acetoxyl groups alpha to a carbonyl group is reported for the first time. The reduction wave of these groups in 20ketosteroids merged with the reduction wave of the carbonyl group. In contrast, two distinct polarographic reduction waves were obtained with 16-hydroxy- (or acetoxy)-l7-ketosteroids.
T
has been no previous report on the direct polarographic reduction of nonconjugated steroidal ketones. HERE
The use of reducible carbonyl derivatives has been reported. Kolfe, Hershberg, and Fieser (8) determined 17ketosteroids polarographically in the form of their Girard derivatives. Girard derivatives of 3-ketosteroids which had failed to reduce under the conditions of Wolfe et al. (8) were polarographed by Prelog and Hafliger ( 5 ) . Other Iv-atersoluble steroid hydrazones were studied by Barnett and Morris (1). Neinian and Markina (4) reported acetone and methyl ethyl ketone reduced directly at -2.20 and -2.25 volts, respectively, in 0.025.T tetramethylammonium iodide solution. I n 0.05-Y tetraethylammonium iodide-75% dioxane, von Stackelberg and Stracke (6) reduced acetone and cyclohexanone a t
-2.46 and -2.45 volts (S.C.E.), respectively. The present work describes the polarographic behavior of ketones in aqueous 90% ethyl alcohol using tetrabutylammonium chloride as base electrolyte. EXPERIMENTAL
Materials. Commercial grade 3-1 alcohol (95% ethyl alcohol-5% methanol) obtained from Publicker Industries, Inc., was sufficiently free from impurities t o be used. Redistillation did not improve its polarographic properties. T h e tetrabutylammonium chloride was polarographic grade. Triton X-100 obtained from Rohm & Haas was used as a maxima suppressor. The steroids surveyed were prepared in the Chemical ReVOL. 31, NO. 6, JUNE 1959
m
1091
T
T 1
4 uo.
. .
- -I---..---------
._ ...’., __ /:., . 9.00
2.00
2.95
2.50
9.75
POTENTIAL, VOLTS
Figure 1. A. E.
Typical polarograms
Pregnenolone 17a-hydroxypregnenolone.
Solution temperatures were maintained constant. ANALYTICAL CHEMISTRY
1 .OmM solution
Table 1. Effect of Concentration on Half-Wave Potential and CurrentConcentration Ratio of Pregnenolone Concn.5 Eli2 id/@ 0,665 -2.44 3.56 3.94 1.33 -2.45 3.95 2.66 -2.44 3.99 -2.43 3.71 3.43 5.34 -2.44 a Millimolar. b Volts os. saturated calomel electrode. c Normalized to drop time of 3.00 seconds, pa. per mmole. RESULTS A N D DISCUSSION
Polarographic Behavior of Typical Ketone. Pregnenolone n-as chosen as a typical nonconjugated ketone. The normal polarographic variables Tyere examined.
20
iH3 c=o
EFFECT O F CONCEKTRSTION ON DIFCURREKT.The diffusion current obtained with pregnenolone was found to be directly dependent upon concentration in only a very limited range (Table I). The half-wave potential was independent of concentration over the range studied. EFFECTOF TEMPERATURE ON DIFFUSION CURRENT. The variation of diffusion current with temperature was determined for pregnenolone from 13’ to 52’ C. The temperature coefficient
FUSIOX
9.75
Polarogram of androstane-3/3,16/3-diol-l7-one
1 SmM solutions
search- and Development Division, Schering Corp., except for the 16hydroxy- (or acetoxy)-17-ketosteroids, which were supplied by the SloanKettering Institute. Apparatus. All the polarograms were run using the Sargent Model X X I recording polarograph. The polarographic cell was a n H-cell containing a normal calomel anode separated from the sample compartment by a n agar plug and a frittedglass disk. The chloride ion of the calomel cell was furnished by a tetrabutylammonium chloride solution. The capillary was a Corning marine barometer tubing with a capillary constant, m2’3 W, equal to 1.811 mg.2’3 sec.-1/2 in 0.1N potassium chloride a t an open circuit. Procedure. Enough steroid was weighed into a 10-ml. volumetric flask t o make the final solution approximately 1.5mM. After the sample had been dissolved in ethyl alcohol, 0.5 ml. of 1 M tetrabutylammonium chloride, 0.1 ml. of 0.27, Triton X-100, and 0.4 ml. of water were added. The solution was made up to 10 ml. Kith additional ethyl alcohol then deaerated with nitrogen for 20 minutes and polarographed. A blank solution containing everything except the sample was polarographed in a similar manner. All the sample polarograms were corrected for the current obtained with the blank. Halfwave potentials, referred to the saturated calomel electrode, and currents were obtained by the line intersection method from these corrected curves. The current-concentration ratios reported have been normalized to a drop time of 3 seconds by means of the relation,
1092
Figure 2. diacetate
9.95 9.50 POTENTIAL, VOLTS
averaged 1.91% per degree with respect to the 25’ C. value. This coefficient is in keeping with a diffusion-controlled process. EFFECT OF MERCURYHEIGHT ON DIFFUSIOKCURREXT. The ratio of the diffusion currents obtained a t two different mercury heights for pregnenolone was 1.29. Ratios of 1.61, 1.27, and 1.00 would be expected for adsorption, diffusion, and kinetic controlled processes, respectively, for the tn o mercury heights employed. REPRODUCIBILITY. The average halfwave reduction potential of pregnenolone was found to be -2.439 volts with a standard deviation (2 sigma) of 0.013 volt. The current-concentration ratios averaged 3.90 pa. per mmole TTith a standard deviation (2 sigma) of 0.08 pa. per mmole (2%). These values are remarkably good considering the poor reduction wave (Figure l), which was usually obtained a t the high negative potentials employed. Survey of Ketonic Compounds. ~ ~ O N O K E T O K E S . T h e polarographic data for the reduction of monoketones are given in Table 11. The half-wave potentials varied from -2.28 to -2.76 volts; most of the compounds mere reduced a t about -2.50 volts. Suclear ketonic groups a t Cat Clt, CI2, and C17 and side chain ketonic groups a t Cz0,CZ2,and Czawere included in this study. The half-wave potential of the ketonic groups increased in the following order: CS < C17. Czo < CIZ < Cil. The steric hindrance factor seems to be operating, as the less sterically hindered ketonic groups are reduced more easily. The current-concentration ratios ( i d / C ) , varied considerably from 2.5 to 4.4pa per mmole. The average was 3.4 pa. per mmole.
Q-HYDROXY AKD a-ACETOXY KETable II. Half-Wave Potentials and Current-Concentration Ratios for Monoketones The reduction of a-hydroxy Carbonyl and a-acetoxy ketones (Table 111) Compound Position El 2 zd/C showed a n enhancement in their i d / c Cholestan-3-one C3 -2 28 4 4 values ; ilb-pregnene-36,21-diol-20-one 17a-Ethyletiocholan-17~-ol-3-one Ct -2 37 3 3 and its 21-acetoxy derivative yielded -2 59 2 7 Cortisone acetate 3,20-biscycloethylene ketal C11 identical i d / C values, n-hich !\-ere about Pregnan-17a-ol-3,11-20-trione 3,20-hiscycloethylene ketal Cl1 - 2 69 2 5 twice the average for a monoketone. -2 76 3 2 Pregnane-3a,2Op-diol-ll-one C11 Three 17-hydroxy-20-ketosteroids were Pregnane-3a,20p-diol-12-one C12 -2 28 3 7 studied ; Ab-pregnene-36, 17 a-diol-20A5-22-1sospirosten-3/3-0~-12-one(gentrogenin) C12 - 2 48 2 9 one. pregnane-3a,lla,l7cu-triol-20-one, 22-Isoallospirostan-30-01- 12-one (hecogenin) Cl? -2 51 2 8 and pregnane-3a,ll~,l7a-triol-20-one Methyl bisnorcholan-3a-ol-l2-one-23-oate C12 - 2 53 3 9 Methyl norcholan-3a-ol-12-one-21-oate Cl? -2 55 3 3 also displayed an enhancement in their Methyl cholan-3a-ol-12-one-25-oate CIZ -2 55 3 0 id,’C values. Two 17,21-dihydroxy-20Androstane-3,17-dione 3-cycloethylene ketal C17 -2.45 3 5 ketosteroids yielded current-concenAj-A4.ndrosten-3p-ol-17-one C17 -2 45 3 7 tration ratios about triple that for a 15-Pregnen-Sp-01-20-one acetate Cro - 2 44 4 1 monoketone. The hydroxyl and aceAb-Pregnen-3P-ol-2O-one (pregnenolone) Czo -2.45 39 toxll groups had a slight tendency t o ~~-2O-Ieonorcholen-3p-ol-22-0ne C22 -2 49 3 8 lower the half-wave potential. A study of five 16-hydroxy (or acet~~-~orcholesten-3p-ol-21-one C24 -2 47 3 5 oxy)-17-ketosteroids (Table 111) revealed that not only was the total i d / C Table 111. Half-Wave Potentials and Current-Concentration Ratios for a-Hydroxy doubled but wave splitting had occurred. (or a-Acetoxy) Ketones A separate and distinct reduction wave Carwas obtained a t about -2.00 volts bonyl followed by the carbonyl reduction wave Hydroxyl PosiCompound Position tion a t -2.45 volts (Figure 2). Eli2 id/P POLYKETOKES. The reduction data A5-Androstene-3p116a-dio1-17- I .99 4 1 of di- and triketones are listed in Table one diacetate CIS C17 -2.44 6 8a IV. The half-wave potentials are sin& Androstane-3p, 16a-diol-7-one lar to those of monoketones, but the -1.99 4 1 diacetate C16 C17 id/C values have increased correspond-2.45 7 3 29 -2.09 ing t o the number of ketonic groups Estrone- 168-01 C16 Cl7 7 Oa -2.46 present in the molecule. 4 @ -1.96 Estrone-16P-01diacetate c 1 6 c 1 7 T\TOKSTEROID.4L KETONES.The re-2.40 7 00 sults of the polarographic reduction of 4ndroetane-3B116p-diol-17-one -2.04 nonsteroidal ketones are compiled in 4 3 diacetate C16 c 1 7 -2.45 7 45 Table V. The straight-chain ketones A5-Pregnene-3p,l7a-diol-20-one C17 were the most difficult to reduce. -2,43 6 4 C20 Pregnane-3a, 11B, 17a-triol-20The half-wave potentials are in the 60 -2.48 (317 cm one same range as in von Stackelberg and Pregnane-Xa, 11a,l7a-tril-20-o Stracke’s paper. The current-concen-2.47 5 9 one C17 C20 tration ratios were similar to those A6-Pregnene-36,21-diol-20-one GI -2 38 7 6 Ceo found for steroidal monoketones. The A5-Pregnene-3B,21-diol-20-one a-hydroxyketone, 3-hydroxy-2-butan-2.37 7 3 21-acetate CZl C?O one, gave one reduction wave similar in Pregnane-Ba, 11p,17a,21-tetrolposition to the reduction wave of 2-2.40 9 8 20-one 21-acetate (217, 21 G O A6-Pree;nene-36,17a,2l-triol 3,butanone, but its id/C was increased 96 -2.35 21-diacetate c17,21 c*o by a factor of 2. It would appear that a Total current-concentration ratio. the reduction potential ( - 1.7 volts) which Kinkel and Proske ( 7 ) reported for 3-hydroxy-2-butanone in 0 . 1 s amTable IV. Half-Wave Potentials and Current-Concentration Ratios for Polyketones monium chloride was due to an elecCarbonyl trolyte interaction rather than to the Compound Position E1 Zdlc compound itself. Androstane-3,17-dione C,, 17 - 2 38 8 0 REACTION MECHANISM.The reducPregnane-X,20-dione C3, - 0 -2 44 7 5 tion of nonconjugated ketones proceeds Pregnan-llp-ol-3,20-dione c,, ?O -2 46 7 4 Pregnan-3p-01- 11’20-dione acetate c 1 1 , -2 46 7 1 through a 2-electron irreversible step, yielding a carbinol as illustrated in C,, 11, 17 -2 37 11 0 Etiocholane-5,11,17-trione c,,11, 20 - 2 41 10 5 Pregnane-3,ll120-trione Equation 1. Comparison of the i d / C values for the nonconjugated ketones Kith that of a conjugated ketone known Table V. Half-Wave Potentials and Current-Concentration Ratios for Nonsteroidal t o reduce through a 1-electron process Ketones ( 2 ) supports this contention. Compound EI,? ad/C Compound E1 2 tdlC Acetone -2 57 5 2 Cyclodecanone -244 49 2-Butanone - 2 59 4 6 Cyclopentadecanone - 2 45 4 7 ‘C=O + 2e + 2H+ --+ ‘CH-OH 2-Heptanone -2 54 5 0 -2 41 3-Hydroxy-Zbutanone - 2 45 8 9 / / Cyclobutanone 1-Acetylcyclohexanol -2 54 8 7 Cyclopentanone 1; 3-Hydroxyl-3-methyl-2Cyclohexanone pentanone - 2 54 10 8 Cycloheptanone -2 48 5 5 When a n a-hydroxy (or an a-acetCyclo-oc tanone -2 43 4 2 Hexane-2,5-dione -2 49 11 6 oxy) group is present, it is reduced prior
TOKES.
~~
.I?
p
VOL. 31, NO. 6 , JUNE 1959
1093
to the reduction of the carbonyl, as shown in Equation 2. -bH
- L H
I
C=O
I
+ 2e + 2 H f +
i:I =O + H20
ACKNOWLEDGMENT
The authors are grateful to Beatrice S. Gallagher, Sloan-Kettering Institute for Cancer Research, for the 16-hydroxy- (or acetoxy)-17-ketosteroids used in this investigation.
(2)
Proof of the structure of the products of large scale reductions will be published (3).
LITERATURE CITED
(1) Barnett, J., Morris, C. J. 0. R., Biochem. J . 40, 450 (1946). ( 2 ) Kabasakalian, P., McGlotten, J., J. Electrochem.Soc. 105,261 (1958). (3) Kabasakalian, P., RIcGlotten, J.,
Yudis, AI. D., J . Am. Chem. SOC. in press. (4) Neiman, &I. B., Markina, Z. V., Zavodskaya Lab. 13, 1174 (1947). (5) Prelog, V., Hafliger, O., Helc. Chim. Acta 32, 2088 (1949). (6) Stackelberg, 31. von, Stracke, W., 2. Elektrochem. 53, 118 (1949). (7) Winkel, A., Proske, G., Ber. 69, 1917 (1936). (8) Wolfe, J. K., Hershberg, E. B., Fieser, L. F., J. Biol. Chem. 136, 653 (1940). RECEIVED for review September 9, 1958. Accepted December 3, 1958.
Coprecipitation of Sodium in Sulfate Determination A Spectrographic Method JOHN
L.
VOTH
Deparfment of Chemistry, University of California, Davis, Calif.
FSpectrographic analysis of sodium offers a new approach to an old problem. In the sulfate precipitation by barium chloride, the coprecipitation of sodium doubles, only, with a 20-fold increase in sodium chloride concentration. Sudden addition of barium chloride solution decreases coprecipitation by half. Coprecipitation reaches a constant value after 6 hours' hot digestion of the precipitate and remains the same over a pH range from 2 to 6. Results indicate that sodium coprecipitation is not so great as reported.
E
in the precipitation of sulfate by barium chloride solution has been attributed to sodium sulfate occlusion (1, 3-5). The spectrograph simplifies the determination of sodium because no further sample preparation is required after precipitation and ashing. 15-aldbauer and Gantz (6) have reported the coprecipitation of ions other than sodium n-here spectrographic techniques Tere used. Allen and Johnston (1) and Johnston and Adams (4) determined the sodium occlusion in barium sulfate precipitation by redissolving the precipitate in concentrated sulfuric acid and reprecipitating by dilution in water. The filtrate was evaporated to dryness and the residue weighed as sodium sulfate. RROR
Reagent grade chemicals were used without further purification. Preliminary spectrographic examination of a barium sulfate precipitate indicated no cations other than barium and sodium. The method of precipitation of Allen and Johnston (1) was closely followed for a comparison of results. The volume of sodium sulfate solution TT as 350 ml., p H 1094 *
ANALYTICAL CHEMISTRY
of precipitation approximately 2, and the weight of the precipitate was 2 grams. The barium chloride solution, aged at least 24 hours (S),was added from a buret with a capillary tip so attached that 21 ml., a slight excess, would be delivered in 4 minutes. Digestion time on the steam bath was 18 hours. The precipitate was filtered through 11-cm. KO.42 Whatman paper. After transfer of the precipitate to the filter paper, it was washed ten times with hot water made acid to methyl orange and then five times with hot water only. The last washing was tested with a 5% silver nitrate solution and to ensure that it had been adequate, two precipitates were further washed with 25 ml. of hot water and the water was tested for sodium ion by flame photometry. Less than 0.3 p.p.m. of sodium, an amount insufficient to affect the results, was present. SPECTROGRAPHY
The precipitates, after ignition, were prepared for spectrochemical analysis by mixing with a buffer composed of one half graphite (National Carbon Co. SP2 grade), one quarter lithium sulfate, and one quarter aluminum sulfate. Equal parts of buffer and barium sulfate were weighed and mixed for 40 seconds on a Wig-L-Bug (Spex Industries, Inc.). Standard samples of the same composition were prepared n-ith reagent grade barium sulfate free of sodium. Sodium sulfate was added to give three samples containing 0.16, 0.064, and 0.025% of sodium, respectively. Triplicate arcings were made on each spectrographic plate of the three samples. The sample electrodes 7.9 mm. in diameter had a cup 4.8 mm. in diameter and 4 mm. deep. A hole was drilled in the bottom of the cup, 1.5 mm. in
I
01
02
I
I
l l l l l l
05
I
I .2
PER CENT
Fiaure 1.
N i 3303.0 Ba 2702.6
Ratio of intensities vs. per cent sodium
diameter, and 8 mm. deep, measured from the bottom of the cup. Twenty milligrams was weighed on filter paper, transferred to the cup, and tamped into the hole a t the bottom with the b u t t end of a 1.5-mm. drill. The cup was then cut away with a lathe, exposing the sample to the arc. The counterelectrode was 1/4-inch rod, 1 inch long with a post cut a t one end 2 mm. in diameter, and 11 mm. long. A direct current arc of 140 volts, 12 amperes, was used with the sample located in the anode. Exposure time \vas 1 minute. A. rotating sector placed before the slit reduced light intensity 507,. An arc gap of 3 mm. was maintained a t a constant position by reference to a magnified image of the arc projected on a screen. The spectrograph was a large Littrow (Bausch RLomb Optical Co.) with quartz prism, photographing the region 2500 to 3500 A. on SA-1 plates (Eastman Kodak Co.). The characteristic curve of the spectroscopic plates was determined from selected lines (2) of the iron arc. The analytical curve was obtained by plot-
