alkyldimethylamines shown in Figure 4 were compared to the results from a chromatogram of a debenzylated sample of the bennalkonium chloride made from this a l k y l d ~ e t h y l a m ~mixture. e Results of this comparison are sh0n.n in Table I1 with the calculated amounts
of benzalkonium chloride which each aminerepresents. LITERATURE CITED
(1 Adams, Re,ed., “Organic Reactions,” VU, p. 278, Wiley, New York, 1963. (2) Schulenberg, J. W., Sterling-Winthrop Research Institute, Rensselaer, N. Y.,
private communication. (3) Southworth, B. C., ANAL. GHEX. 28, 1611 (19561. (4) U. S. Pharmacopeia, 16th Rev,, p. 82, The United States Pharmacopeia Convention, Inc., 1960. RECEIVED for review July 3, 1961. Accepted October 9, 1961.
PETER E. MANNII’ and JOSEPH E. SlNSHElMERl Colleges o f Pharmacy, University of Rho& Island, Kingsfon, Is. 9., and The Yniversify o f Michigan, Ann Arbor, Mich. Variously substituted hydroxytones were studied, to determin whether differences could be detecte in their rate of reaction with blue tetrazolium. In many cases substantial erences were found which could be related to either the alpha or beta i group with position of the h respect to the keto or to whether the hydroxyl group i s primary, secondary, or tertiary. A correlation of the electron-donating ability of substituent groups with formazan development is indicated. Results also suggest the possibility of assaying for individual ketols and mixtures of ketols. Three compounds of medicinal interest tetracycline hydrochloride, alloxantin dihydrate, and erythromycin base prove reactive toward blue tetrazolium, and their quantitative determination via a “tetrazolium reactionlu seems feasible.
-
EYER and Lindberg (8) as well as
Rosenkrantz (11) have demonstrated the usefulness of rate studies involving blue tetrazolium for the structure elucidation of Ac3-ketosteroids and catecholamines, respectively. As a consequence of their investigation of steroids, Meyer and Lindberg (8) established that increased reactivity is to be expected from those compounds which contain an a-keto1 moiety. However, no data were elicited to indicate the effects of varying substituents upon reactivity of the ketol grouping. Therefore, it seemed worthwhile to investigate the rate of reaction of model compounds with blue tetrazolium. The information gleaned from such studies is useful not only as a tool for structure elucidation, but also for the quantitative analysis of individual ketols and of ketol mixtures. 1 Present addrew, College of Pharmac University of Michigan, Ann Arbor, Mi&
1900
*
ANALYTICAL CHEMISTRY
TIME (MIN.1
Figure 1. Rate of formazan formation for 1 X 1 O-b solutions of a-hydroxyketones A. Dihydroxyacetone (reeorded as monomer) B. 3-Hydroxy-?-butanone C.
Hydroxypropanone
EXPERIMENTAL
All spectrophotometric measurements were made with a Beckman Model DU spectrophotometer. All melting points are corrected. Unless otherwise specified, reagents and test compounds may be obtained from Eastman Organic Chemicals. Reagents. Blue tetrazolium (Dajac Laboratories), A commercial sample was purified by recrystallization from 95% ethyl alcohol-anhydrous ether according to the method of Weichselbaum and Margraf (13). Purification was continued until a portion of the salt in absolute ethyl alcohol showed a constant absorbance a t 254 mh.
Absolute ethyl alcohol. Following distillation from 2,4-dinitrophenylhydrazine (10 grams per liter) the alcohol was dried by treatment m t h sodium and diethyl phthalate as described by Fieser (4). It is necessav to keep the aqueous concentration of reagents a t a minimum because water has been shown to inhibit the reaction (0) *
Tetramethylammonium
hydrodde.
A 0.03OON solution was prepared by dilution of a 10% aqueous solution with absolute ethyl alcohol. Test Compounds. Solids were recrystallized to constant melting point. Li uids were fractionally distilled un%er nitrogen. Significant disagree-
0.301
I .2 I_I
/
O
m a
O F '
"
20
"
40
'
60
"
80
'
"
100
"
TIME (MIN.)
Figure 2. Rate of formazan formation for 1 solutions of a-hydroxyketones
0 2
X 10-6M
!
0
A. or-Hydroxyacetophenone 8. a-Pyridoln C. Benzoin D. Furoin E. Anisoin
ment with published values was obtained in two cases: hydroxypropanone ny (obsd.) 1.4189, n': (lit.) 1.4295 (6) and 4-hydroxy-3-methyl-2butanone n2,0 (obsd.) 1.4300, ny (lit.) 1.4340 (1%). U.S.P. reference standard cortisone acetate and U.S.P. reference standard tetracycline hydrochloride were used as supplied. Ascorbic acid, fructose, rhamnose, and erythromycin base (Abbott Laboratories) were also used without further treatment. Dihydroxyacetone dimer (Wallerstein Co.). Purification of the commercial product was conducted according to the method of Reeves and Renbom (10) as modified by Bell and Baughan (1). The absence of methylglyoxal was ascertained from the procedure of Wolfrom and Arsenault (14). Procedure. To each of 12 amber bottles, appropriately labeled as to the time of reaction, was added 2 ml. of a n absolute ethyl alcohol solution of test compound. The concentration of test compound in the reaction mixture was approximately t h a t represented in Figures 1 to 5. The bottles were allowed to equilibrate a t 30" i= 0.1"in a water bath for 10 to 15 minutes, after which 2 ml. of a 0.1% absolute ethyl alcohol solution of blue tetrazolium was added, immediately followed by 2 ml. of 0.0300N tetramethylammonium hydroxide. The mixture was allowed to react under these conditions for the prescribed time, when 1 ml. of alcohol-diluted hydrochloric acid (prepared by diluting commercial 37% C.P. hydrochloric acid 10 times with absolute ethyl alcohol) was added to stop the reaction. The solution was allowed to reach room temperature, and its absorbance against air determined a t 530 m p A blank was prepared by mixing 2 ml. of 0.1% blue tetrazolium solution,
20
40
60
60
100
T I M E (MIN.)
Figure 3. Rate of formazan formation for 1 solutions of a-and P-hydroxyketones
X 1 0-2 M
(3-Hydroxyketoner A. 4-Hydroxy-2-pentanone C. 4-Hydroxy-3-methyl-2-butanane E. 4-Hydroxy-4-methyl-2-pentanone a - H ydroxyketoner E. 2-Hydroxy-3-methyC2-cyclopentene- 1 -one D. 3-Hydroxyflavone
2 ml. of 0.0300N tetramethylammonium hydroxide, and 2 ml. of absolute ethyl alcohol. The absorbance was determined a t times corresponding to that of the reaction mixture, the only difference being that the blank was a t room temperature for the entire time. Compounds which yield colored solutions without blue tetrazolium under the reaction conditions (tetracycline hydrochloride, furoin, and a-pyridoin) necessitate the preparation of a second blank which consists of 2 ml. of absolute ethyl alcohol, base, and a solution of test compound, respectively. Deviation from the above procedure was made in several instances. Maltol (3-hydroxy-2-methyl- 1,4-pyrone), 4hydroxy-2-pentanone, 3-hydroxdavone, and 3-hydroxy-3-methyl-2-butanone were tested by measuring the absorbance of the reaction mixture in the same manner as the blank. These samples were not treated with mid, because the rate of reaction is slow enough to permit accurate determination of absorbance without stopping the reaction. Absorbance values were obtained in the following manner: (Absorbance of reaction mixture) 6/7 (absorbance of blank) except where the reaction was not stopped by acid. In the latter case the net difference between absorbance values of reaction mixture and blank was used. These values were adjusted t o correspond to an appropriate con-
Table 1. Formazan Formation Model Compounds with Time
Compound
by
Absorbance At120 min. min. At 6
1 x 10-6M Concentration 0.475 Dihydroxyacetone4 0.447 3-Hydroxy-2-butanone 0.302 0.019 0,141 H droxypropanoneb 0.124 3- ydroxy-3-methyl0.000 2-butanoneb 0.000 crHsdroxyacet ophe 0.213 0.304 none a-Pyridoinc 0.269 0.174 0.262 Benzoin 0.158 0.228 0.180 Furoin 0.214 Anisoina 0.046 0.000 Methylbenzoind 0.000
H
1 x 10-*;M Concentration PHydroxv-2-pentanoneb" 0.056 1.364 4-Hydroxy-3-methyl2-butanone'~ 0.027 0.730 4-Hydroxy-4-methyl2-pentanone 0.003 0.063 3-Hydroxyflavone 0.108 0.188 2-Hydroxy-3-methyl2-cyclopenten-l-onea 0.026 0.771 Maltol(3-hydroxy-2methyl-l,4-pyr0ne)~ 0.000 0.000 0 Available as dimer from Wallerstein Co., calculated as monomer. b K & K Laboratories. 0 Aldrich Chemical Co. d Prepared in this laboratory. * Dow Chemical Go.
VOL. 33, NO. 13, DECEMBER 1961
1901
0,42t
nJ
I
#
03t
E 024 0
m i Q
0.1s
20
40
60
80
100
0
20
TIME(MIN)
d Qorrnazanformation for I X 1 Q + M
DISCUSSION
Earlier work (8) has indicated that the “tetrazolium reaction” is more specific for primary a-hydroxyketones Then low base concentration and temperature are employed. Our results confirm this observation. Thus, the greatest difference in formazan production between the primary ketol, hydroxypropanone, as compared to the secondary compound, 3-hydroxy-2-butanone1 occurs within the first 5 minutes (Figure 1). Also, a-hydroxyacetophenone initially shows greater formazan production than either 3-hydroxy-2-butanone or any of the four secondary aromatic ketols examined (Figures 1 and 2). Dihydroxyacetone with two primary hydroxyl groups shows the most reactivity (Figure 1) The tertiary alcohols, methylbenzoin at and 3-hydroxy-3-methyl-2-butanone, 1 X 10-6fW concentration fail to react within 2 hours. Even a t concentrations of 2.96 X and 3.5 X 10-2LM, respectively, they produce an absorbance no greater than 0.038 after 2 hours.
Figwre 5.
noticeable reaction within 2 hours (Figure 3). Under the conditions employed 8hydroxyketones in 1 X 10-6M concentration fail to react within 2 hours. However, in concentrations approaching 1 x 10-2M 8-hydroxyketones produce significant reaction (Figure 3), although a plateau, which would indicate complete reaction, is not seen in any of the cases studied. Members of the aromatic series studied (Figure 2) offer an opportunity to evaluate electronic effects upon the reaction. Results suggest that the amount of formazan produced after 2 hours is influenced by the character of substituent groups. I n order of increasing formamn production the series anisoin, furoin, benzoin, and a-pyridoin can be formulated. The electron-donating ability of the substituent rings has been proposed by Callowoy (2)t o be:
I
Compounds of the type -C-C=C-
d
1
bH (maltol, 2 - hydroxy - 3 - methyl 2cyclopenten - 1- one, and 3 - hydroxyflavone) also do not react at 1 X 1Q-6.W. However, with the exception of maltol, a n increase in concentration produces a e
100
Rate of Qormazanformation
Rhamnose monohydrate Cortisone acetate
centration and recorded in Table I and Figures 1 to 5.
1902
80
A. 6.63 X 10-’M erythromycin base B. 1.79 X 1 0-*M tetracycline. HCI C. 2.17 X 10-4M alloxontin dihydrate
A. Fruefose 8. Ascorblc acid D.
60
TIME ( M I N )
sdutions C.
40
ANALYTICAL CHEMISTRY
-
p-Methoxyphenyl was not included in Calloway’s study, but it is well known that resonance effects operate t o in-crease the electron density about the carbon para t o the methoxy group. Correlation of reactivity with the electronic character of substituent groups is most apparent when compounds with the same ring system are compared as represented by benzoin and anisoin. Differenkes are noted too,
when methyl is replaced by phenyl, as illustrated by a-hydroxyecetophenone and hydro.xypropanone. I n both cases there is a difference not only in the amount of formazan produced, but also in the initial rates of reaction. It would seem that the number of hydrogens on the carbon bearing the hydroxyl group is more important in influencing. degree of reactivity than electronic effects, for a-hydroxyacetophenone is more reactive than any other aromatically substituted a-keto1 (Figure 2). This is most evident when ahydroxyacetophenone is compared to methylbenzoin. To correlate results obtained using our procedure with those described elsewhere (8, 7) the reactions of cortisone acetate, fructose, and ascorbic acid (Figure 4) were investigated. In general, this procedure results in reactions which are slower than those previously reported. The a-hydroxyaldehyde, rhamnose monohydrate] is less reactive than the a-ketol, fructose (Figure 4). Erythromycin base, tetracycline hydrochloride, and alloxantin dihydrate a t 1 X 1O-sIM concentration react in accord with the model compounds they resemble (tertiary alcohols, /%hydroxyketones). Greater concentrations of these compounds produce definite reaction (Figure 5), suggesting that methods of assay employing blue tetrazolium can be developed. A final application would be the quantitative determination of a reactive compound in the presence of a less reactive compound--e.g., a mixture
of a n a- with a p-hydrosyketone. An approach similar to that employed by Izzo, Keutmann, and Burton (6) may be suitable. LITERATURE CITED
(1) Bell, R. P,, Baughan, E. C., J . Chem. S O C . 1937,1947. (2) Calloway, N. O., Chem. Revs. 17, 327 (1935). (3) Fairbridge, R. A., Tyillis, K. J., Booth, R. G., Biochem. J. 49, 423 (1951).
(4) Fieaer, L. F., “Experiments in Organic Cheniistry,” 3rd ed., p. 286, D. C. Heath, Boston, 1957. (5) Izzo, A. J., Keutmann, E. € Burton, I.,
R. B., J . Clin. Endocrinol. and Metabolism, 17, 889 (1957). (6) Kling, A., Ann. chim. et phys. [SI 5,471 (1905).
(7) Mader, W. J., Buck, R. R., ANAL. CHEM.24,666 (1962). (8) Meyer, A. S., Lindberg, M. C., Ibid., 27, 813 (1955). (9) Recknagel, R. O., Litterin, M., J . Lab. Clin. Med. 48, 463 (1956). (lO).ReeveP, H. G., Renbom, E. T., Bzochem. J . 25, 411 (1931).
( 3 1 ) Rosenkrantz, H., Arch. Biochem. Biophys. 81, 194 (1959). (12) (12)Wagner, R. B., J. Am. Chem. SOC. 71, 3214 (1949). (13) Weichselbaum, T. E., Margraf, K., J. C clin. h . Endocrind. and Metabolism 15,970 (1955). (14) Wolfroin, hI. L., Arsenauit, @. P., J . Ora. Chem. 25.205 (1960). R E C E I Yfor ~ rekev X a y 29, 1961. Accepted August 21, 1961. Ahetrncted in
part from a thesis submitted Graduate School, University of Island, by Peter E. Blanni in fulfillment of the requirements mnster of science degree.
to the Rhode partial of the
A System far Identification of Barbiturates in GEORGE W. STEVENSON Department o f Pharmacology and Toxicology, Sckool of Medicine, University o f California, 1os Angeles 24, Calif.
b A previously described butyl etheraqueous partition procedure is combined with two methods of permangana-te oxidation and two of alkaline hydrolysis for systematic identification of barbiturates in blood. The partition procedure divides barbiturates into nonpolar and polar classes. The nonpolar class is divided into saturated and unsaturated b y KMnOa oxidation. Identification of the saturated is completed b y hydrolysis in hot alkali, and of unsaturaled b y washing their chlaroform solutions with KMn04. Polar barbiturates are characterized b y rate of alkaline hydrolysis a t 25” C. The alkali-resistant polar barbiturates are identified b y washin form solutions with KMnQa. Components of mixtures can also b e identified and their proportions determined. Each method is based upon spectrophotometric measurement of decrease of barbiturate absorb a nce. barbiturate identification methods are not readily applicable to blood or tissue samples, since small qumtities(25 t o 250 pg. in 5 ml. of blood), their occurrence in mixtures rather than singly, and the presence of many other compounds pose special problems. Although x-ray diffraction is the most specific of all techniques, solution methods are preferred. Mixtures, need for isolation, and polymorphism limit use of procedures requiring crystals. The procedure of Goldbaum (6) based on small differences of ultraviolet absorption is the most rapid, but identification is not positive because each barbiturate does not have ti characteristic curve, mixtures cannot be detected, and curves are sensitive to ultraviolet-absorbing impurities. Barbiturates on paper chromatograms OST
can be located by ultraviolet lamp and their presence confirmed by application of the Goldbaum spectrophotometric proceduie to the eluate (6). Unsaturated barbiturates can be detected by their permanganate decolorization (2, 3, 10) and bromobarbiturates by their debromination ( 2 ) . Identification of each of thg many barbiturates used in medicine has not- been possible using these methods because of failure to resolve or distinguish barbiturates of similar polarities and the limited specificity of the ancillary techniques. An advantagc of spectrophotometric procedures i? that they measure barbiturate directly, not indirectly by the disappearance of a reagent such as permanganate, which is assumed to be reactifig with barbiturate. Analyses depending on the removal or destruction of compounds measured spectrophotometrically can be termed disappearance spectrophoton;etry. Broughton’s (1) and Curry’s (4) procedures and those described in this paper are of this type. Broughton’s procedure characterizes barbiturates by their rates of alkaline hydrolysis; Curry’s by reaction with concentrated sulfuric acid. Though these are of clinical value, they characterize classes rather than single barbiturates and must be combined with other techniques such as paper chromatography for positive identification. With the aim of developing a system for positive identification of barbiturates, their liquid-liquid partition behavior was investigated in this laboratory. Chloroform (aqueous) and butyl ether (aqueous) partition coefficients of the available barbiturates have been tabulated (9). Reference has already been made to existing partition data (8). A system for preliminary identification was developed in which a butyl ether solution of the barbiturate was washed
successively with two portions of p H 9 buffer and one of liV sodium hydroxide (8). As with paper chromatography, some compounds of sinular polarities are not distinguished and certain mixtures not detected. Two permanganate oxidation techniques and two alkaline hydrolysis procedures systematically performed on the partially resolved fractions from the partition procedure complete the identification system shown in Figure 1. APPARATUS AND REAGENTS
Apparatus used ( 7 , 8 )and preparation of butyl ether and several other reagents (7) have been described. CHLOROFORM.R a s h reagent grade (Merck 50-pound drurng can be used) with several 0.1 volumes of 0.5 to 1N MaOW and finally nith distilled water. Store in glass-stoppered bottles. If this is to be stored more than a week, overlay it nith a 2-cm. deep layer of 1M sodium sulfite and keep in the dark. (Usable for a t least a year.) SATURATED KMnQ4. Heat 90 granis of KMn04 with 1 liter of distilled water on a steam bath for an hour. Allow to stand several daw. Store in the dark. (2.41N if stored a t 25’ C.) KMnO., WASHSOLUTION. To 10 ml. of 0.5M -phosphate buffer and 14.6 meq. (6 ml. of 2.41N) of K(MnO4 solution add distilled water up to 100 ml. 0.5M PHOSPHATE BUFFER. D i S S O h ? 34.0 grams of reagent grade potassium dihydrogen phosphate and 36.5 grams of reagent grade anhydrous disodium hydrogen phosphate in sufficient water to make 1 liter of solution. (Tenfold concentrated KBS pM rji% phosphate standard buffer.) PROCEDURE
The procedure belo%-ispreceded hy ti:@ prelimhary identification method (8) and uses NaOH 1 and Borax 1 fracliom therefrom. If the value of Ratio 1 la
