Determination of Meprobamate in Urine Applicability to Other Compounds Containing an N-H Group GORDON H. ELLIS and CHARLES A. HETZEL Wyeth Institute for Medical Research, Radnor, Pa.
b A colorimetric micromethod for water-soluble compounds containing an N-H group is described. The compound reacts at room temperature with a hypochlorite solution at a pH of 10.5 to form an “active” chlorine derivative. The excess hypochlorite is decomposed by phenol in dilute hydrochloric acid solution and the “active” compound then reacts with an excess of potassium iodide. The iodine formed is measured either as potassium triiodide or as the starchiodine blue complex. The application of this procedure to the determination of meprobamate in urine is described.
mended that they be made up just before use. Phenol Solution. A 0.5% C.P. phenol in 0.1N hydrochloric acid. Starch Solution, 1%. Suspend 1 gram of indicator grade starch in a little cold water and pour this into the remainder of the water at a boil. Remove heat and allow to cool. Starch-Potassium Iodide Solution. Dilute a n equal volume of 1% starch and 1% potassium iodide solutions with sufficient water to bring the concentration of each constituent to approximately 0.033%. This should be done just prior to use. PROCEDURE
compounds containing a n 3-H group react with a chlorinating agent such as chlorine or sodium hypochlorite to form S-chloro derivatives. These act as oxidizing agents and their concentration can be estimated by measuring the amount of iodine liberated by their reaction with potassium iodide. Rydon and Smith (3) have described a method, based on this principle, for detecting amino acids on paper following chromatography. Pan and Dutcher ( I ) have given a method for detecting acetylated neomycins in a similar situation. I n adapting the principle outlined above to a quantitative micromethod, the compound the authors studied most extensively was meprobamate (2niethyl-2-n-propyl-l,3-propanedioldicarbamate). ANY
REAGENTS
Borate Buffer, 0.05M. For 1 liter use 3.1 grams of C.P. boric acid, 3.7 grams of C.P. potassium chloride, and sufficient sodium hydroxide to bring the p H to 10.5 (glass electrode). The potassium chloride serves to increase the stability of the hypochlorite solutions made from this buffer. Chlorinating Solution. A 5.25% sodium hypochlorite solution (Chlorox) is diluted 1:30 with 0.05JI borate buffer. Calcium hypochlorite may also be used. For calcium hypochlorite containing 74% of available chlorine use a 0.09% solution in the same buffer. Although these dilute hypochlorite solutions are relatively stable over a period of several hours it is recom1090
ANALYTICAL CHEMISTRY
The determination is carried out in 16 X 150 mm. test tubes as follon-s. T o meprobamate standards in 1 ml. of water, to the unknown in the same volume of water, and to a blank of 1 ml. of water, add 1 ml. of the chlorinating solution, taking care not to touch the sides of the tube with the delivery end of the pipet. Shake gently to mix and keep a t room temperature for 15 minutes. Then add 1 ml. of the phenol solution rinsing don n the rralls of the bottom half of the tube, and shake gently. The acid in the phenol solution brings the p H to approximately 3 a t which acidity the excess hypochlorite is rapidly decomposed by phenol. The choice of phenol for this purpose is based on the findings of Pryde and Soper ( 2 ) . At this point the chlorine derivatire of the meprobamate or other K-Hcontaining compound may also decompose. Because the reaction between phenol and hypochlorite is essentially complete in 3 to 4 minutes, a time interval of 5 minutes was chosen. At this time, add 3 ml. of 0.3% potassium iodide solution in n t e r and 5 minutes later determine the per cent transmission a t 350 mp, using water as the reference. For a more sensitive procedure, add 3 ml. of the starch-potassium iodide solution and find the per cent transmittance a t 625 mp. Convert the readings to optical density units and substract the absorbance of the blank from all readings. Find the absorbance for a convenient unit of the standard and calculate the concentration of meprobamate in the unknown. A Bausch & Lomb Spectronic 20 colorimeter was used. but a n y good filter colorimeter would be suitable.
DISCUSSION
K h e n the potassium triiodide color a t 350 mp is used, the absorbance for 10 y of meprobamate ranges between 0.12 and 0.15 and Beer’s law holds fairly Re11 up to 30 y per tube. The method is approximately tm-ice as sensitive when using the starch-iodine blue color. Because the concentration is not always strictly linearly proportional to absorbance, it is advisable to run three standards of 10, 20, and 30 y when the potassium triiodide color is used and 5, 10, and 20 y when the starch-iodine color is used. If the calibration curve is not straight use the standard closest to the unknown for calculating the results. If many samples are to be run, itris helpful to use a colorimeter m-hich can be operated quickly since there is the limiting time of 5 minutes during which a set of standards and samples must be run. The authors have found that samples can be started a t intervals of 20 seconds; hence, a set of 15 can be run a t one time. At some slight sacrifice in sensitivity and adherence to Beer‘s law the time alloved for the phenol-hypochlorite reaction can be extended to 6 to 8 minutes and the same time interwl used before reading the samples in the colorimeter. The theoretical amount of iodine formed can be calculated if one assumes that: the initial reaction goes to conipletion, one C1 links to each N originally linked to either one or two hydrogens, this linkage is stable during the reaction with phenol, each x-Cl group liberates two iodine from potassium iodide in the final reaction. To obtain an over-all estimate of the extent to M hich these assumptions are valid a calibration curve for the colorimeter a t 350 mp n-as prepared using standard solutions of iodine in potassium iodide, and from this n-as calculated the amount of iodine formed n-hen various compounds were used. The actual amount found expressed as a percentage of the theoretical amount and called “per cent of completeness” serves as a n estimate of the suitability of the method for use with various compounds. The data shown in Table I indicate that this method is applicable to a variety of water-soluble compounds containing an N-H linkage.
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. T h e steroids surveyed were prepared in the Chemical ReVOL. 31, NO. 6, JUNE 1959
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