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 b y 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 i t 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 K M n 0 4 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 rji36 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
1. Extract with 2. Extract with 3. Extract with
NaOH 1
NONPOLAR (I) (Ratio 1 5 1.5)
I/, I/,
vol. of pH 9 buffer (Borax 1) vol. of pH 9 buffer (Borax 2) vol. of IN NaOH (NaOH 1)
Borax 1
POLAR (11) (Ratio 1 > 1.5; Ratio 2 > 1.7) 1 ml. Borax 1 100 pl. 8N NaOH
KMnO, oxidation
+
(partially destroyed)
(destroyed)
Identification by:
Saturated (IA) Ratio 1. Alkaline hydrolysis (100' C., 15 min.)
Unsaturated (IB) (Extract fresh blood sample with CHCls) Ratio 1. ICMnO, wash of barbituric acid in CHCls Figure
1.5 or less, follow the procedure for nonpolar barbiturates. If i t is greater than 1.6, follow the procedure for polar barbiturates. Nonpolar Barbiturates (Class I). D E T E C T I O N O F UNSaTURATED BARBITURATE.Pipet 3 ml. of N a O H 1 into a 13-mi. centrifuge tube and add 100 pl. of saturated KMnOd. After mixing, let stand for 5 minutes. Add solid sodium bisulfite until the purple color disappears and is replaced by a brown precipitate of MnOe. Add 300 pl. of 7 M HsP04 and 5 ml. of butyl ether. Equilibrate layers by rapidly inverting the tubes 100 times. Centrifuge. Remove and discard the aqueous phase by pipet. Its p H should be 6 or lower. Decant into a clean tube. Using 5 ml. of butyl ether in another tube as a blank, add 3 ml. of 1N NaOH to each. Equilibrate the layers, centrifuge, and transfer 1ml. of the sodium hydroxide (NaOH 2) to an absorption cell. Measure the amount of barbiturate present as in the total barbiturate procedure. The AAzao(o-h) is thus obtained. Calculate the fraction of unsaturated barbiturate present in K'aOH 1 from Equation 1. SATURATED BARBITURATE (CLASSIA). If the P A z ~ ~ indicates ( ~ - ~ ) the presence of saturated barbiturate] transfer about 1 ml. of the KaOH 2 and 1 ml. of the blank to 5-ml. glass-stoppered borosilicate volumetric flasks. Heat them on a steam bath for exactly 15 minutes. (The stems of the flasks must be shielded from the steam t o avoid evaporation of solution. In this laboratory the following arrangement has enabled the simultaneous treatment of up to 10 samples: Stem-size holes were cut in rubber sheet which was pulled down over the volumetrios supported on a wire 1904
ID
ANALYTICAL CHEMISTRY
+ Alkali-Labile (IIB)
$lkali-Resistant (IIA) Ratio 2. KMnO, wash of barbituric acid in CHClr; also KMnOl oxidation to distinguish cyclobarbital and probarbital
Ratio 2. Measurement of rate of disappearance of barbiturate in alkali at room temperature
1. System for Barbiturate Identification
ings on the above alkalinized Borax 1 gauze, and the rubber edge was sealed within 5 minutes to detect methyldown with a cork ring and ring clamp.) phenyl barbituric acid. Repeat the Cool rapidly. Decant solution and readings on the solutions kept in closed blank into cells and read the absorbcontainers at 24" to 28" C. for 1 to 2 ances at 260 (&(), 255, and 250 mp. hours to detect Dial and allylphenyl Calculate the per cent of barbiturate barbituric acid. If the Ratio 2 value remaining using Equation 2. When (8)is 3 or less and no appreciable loss of amobarbital and pentobarbital are the absorbance has occurred within 2 hours, only saturated barbiturates present, an alkali-resistant barbiturate is present. calculate their proportions using EquaIf within 2 hours absorbance has detions 2, 3, and 4. UNSATURATED BARBITURATE(CLASS creased at the rate of approximately 2% per hour and the Ratio 2 value is in IB). Extract another 5-ml. portion the vicinity of 4, the presence of phenoof the blood sample with 25 ml. of barbital is indicated. This should be chloroform. Upon 5 ml. of the chloroconfirmed by repeating the reading at form in a 13-ml. tube carefully layer about 8 hours. If the Ratio 2 value 1 ml. of KMnOd wash solution. Invert indicates the possibility of barbital, the tube approximately 160 times another reading should be made at during a period of exactly 1 minute. about 24 hours. Centrifuge and take off the permangaALKALI-RESISTSNT BARBITURaTES nate immediately. Decant the chloro(CLASS IIA). Pipet 3 ml. of Borax 1 form into a clean tube. Extract this into a tube. Add 30 pl. of 7 M HaPo4 chloroform, another 5-ml, portion of the and 15 ml. of chloroform. Equilibrate chloroform extract, and 5 ml. of chlorolayers and centrifuge. Remove the form for a blank with 1.25 ml. of I N aqueous layer, Pipet 5 ml. of additional NaOH. Determine the absorbances a t chloroform into one tube for a blank, 260 mp before and after the addition of and S m l . portions of the chloroform 250 pl. of 2.62A4 ethylenediamine. extract into each of two other tubes. 2HC1. Calculate the per cent of barThen carry out the KMn04 wash probiturate remaining using Equation 5. cedure as with the Class IB barbi(KMn04 Wash), turates. Calculate the per cent of Polar Barbiturates (Class 11). Measbarbiturate remaining using Equation 5. ure the absorbance of 1 ml. of Cyclobarbital and probarbital, whose Borax 1 os. blank Borax 1 at 260 per cent remaining values are close to mp before and immediately after the 100, are distinguished by a KRlnOd addition of 100 pl. of 8N NaOH. If oxidation procedure, Combine the recloudiness develops on addition of alkali, maining Borax 1 solution (approxicontacting the liquid with air clears it. mately 1 ml.) and the 1.1 ml. of alkaRemove the liquid into the 1000-p1. linized Borax 1 used in the alkaline pipet used for transferring and mixing hydrolysis a t room temperature above. and then expel i t through the air into Carry out the oxidation given under the cell. Repeat several times until Detection of Cnsaturated Barbiturate, clear. ALKALI-LABILE BARBITURATES but extract the butyl ether extract with 1.25 ml. rather than 3 ml. of 1N NaOH. (CLASSIIB). Repeat the 260-mr read-
Absence of barbiturate absorbance indicates cyclobarbital; absorbance indicates the presence of probarbital. Polar Barbiturates in the Presence of Nonpolar. Remove t h e nonpolar barbiturates from Borax 1 b y butyl ether washing. To 4-ml. portions of Borax 1 add 4-ml. portions of butyl ether. Equilibrate the layers, remove the butyl ether, and repeat the process twice more. Then process the washed Borax 1 as under Polar Barbiturates (Class 11). The per cent of each of the barbiturates which remains in the borax solution is given in Table I.
Table 1.
Ratio 1
I. Nonpolar A. Saturated CALCULATIONS Hexethal Pentobarbital Fraction of unsaturated barbiturate = Amobarbital 1 - 1.05 A A ~ ~ O ( ~ ~ ~ / A A ~ (1) B O ( G . - ~ ) Butabarbital Butethal Hydrolysis a t 100' C. B. Unsaturated Sigmodal % barbiturate remaining = Secobarbital 100 A2601/A?601 (2) Butallylonal Allyl see-butyl For mixtures of amobarbital and Allyl isobutyl pentobarbital: Cycloheptenyl ethyl Proportion of amobarbital = Nostal (96 - % remaining)/53 (3) 11. Polar where 96 = Cr, of pure pentobarbital A. Alkali-resistant remaining and 53 = difference between Aprobarbital % pure pentobarbital remaining and Cyclobarbital 70 pure amobarbital remaining (96 - 43). Vinbarbital Probarbital Proportion of pentobarbital = B. Alkali-labile 1 - (proportion of amobarbital) (4) Allyl phenyl Dial For identification of Class IB: Phenobarbital Methyl phenyl % barbiturate remaining = Barbital 100 ( A.qZ60) oxidized (A.4260) unoxidized
Data for Identification of Barbiturates
Ratio 2
Alkali Hydrolysis % Rates of .~ remain. destruction, 15 min., %/hour, 100' C. 25-28' C.
Borax Buffer Washed 3 Times with Equal VOl. of Butyl KMnQ, Ether, Wash % Bar% biturate Remain- Remain1ng
1%
0.10
40
1
0.25 0.33
96
4
43 96 44
30
0.87 0.86
1.50 1.51
6
30 93 68 87 36
0.12 0.15
23
1 9 16 29
71 68
38 40
2.0
1
2.5
84 36
53 64 67
92
70
0.40 0.51 0.93
1.52
1.19
1.6 1.7
1.44 2.0
1
47
2.6 2.7 3.0
3.5 4.4
4.1 8.0 1.7
7.9
2%/min.
7.9
0.8
70 75 84 88 91
(5)
DISCUSSION
The division of barbiturates into nonpolar and polar classes is arbitrary but convenient. Two types of barbiturates, the thio and N-methyl which belong on the basis of partition coefficients to the nonpolar clam, are not considered, since they have distinctive ultraviolet absorption properties in strong alkali, the former with peaks at 305 nip, the latter a t 245 mp. When Ratio 1 values approximate 1.5 and Ratio 2 values 1.7, properties of the contiguous members of each class must be compared with those of the unknown. Data given in Table I are not complete, because only data necessary or useful for identification are included. Most efficient determination of secobarbital, pentobarbital, and amobarbital, most common ingredients of nonpolar barbiturate mixtures, decided the order of performance of the tests. Potassium permanganate removal of unsaturated barbiturate is complete, and if a mixture of pentobarbital and amobarbital remains, the proportionate decrease in absorbance due t o hydrolysis of amobarbital is greater after removal of secobarbital, the only common unsaturated barbiturate. The only unsaturated barbiturate of lower or comparable polarity is sigmodal, which has a
higher absorbance in'lN sodium hydroxide a t 260 mp than at 255 mfi, like the p-bromallyl barbiturates, butallylonal and Nostal, and unlike the other barbiturates. Secobarbital and sigmodal can be differentiated also by the KMn04 wash. If Ratio 1and unsaturation suggest allyl isobutyl barbituric acid, hot alkaline hydrolysis can be used to confirm it, since it is the only alkalilabile member of Class IB. Polar barbiturates are better divided into classes by alkali resistance than by unsaturation. Hot alkaline hydrolysis cannot distinguish the more susceptible ones because of too rapid destruction. Hydrolysis a t room temperature requires several readings but the procedure requires little time, and together with Ratio 2 values readily identifies the alkali-labile compounds. The alkaliresistant compounds are readily distinguished using the Killn04 wash except for cyclobarbital and probarbital. Saturated probarbital is readily distinguished from unsaturated cyclobarbital by permanganate oxidation in aqueous solution. Methods of analysis of mixtures other than those of secobarbital, pentobarbital, and amobarbital described above or of nonpolar and polar barbiturates using the partition method
alone (8) can be devised, using the data in Table I. An advantage of these procedures is that proportions of components of any of the mixtures are accurately determined-for example, of mixtures containing each of the four common barbiturates. This is apparently not possible with other procedures. Further extractions of the various barbiturate fractions may be used to separate barbiturates of differing polarities, as in the removal of nonpolar barbiturates by butyl ether from the polar barbiturates in borax buffer, data for which are given in Table I. The permanganate oxidation techniques used are dependent on oxidation of unsaturated barbiturates to polar diols which have unaltered chromophores but are not extractable into butyl ether or chloroform from water solutions. These are specific for unsaturated barbiturate as compared with the nonspecific disappearance of permanganate color seen with many other compounds. Oxidation in homogeneous aqueous solution is not useful for differentiation of the various unsaturated barbiturates because of the rapid rates of these reactions. These rates were don-ed by washing a solution of the barbituric acid in chloroform with VOL. 33, NO 13, DECEMBER 1961
1905
permanganate. Standard conditions used enabled a reproducibility within &5% of the tabulated values. Permanganate solutions in distilled water did not give reliable results. Since this was probably due to inadequate p H buffering, a permanganate reagent Q.05144 in NBS phosphate buffer was used thereafter. Values reflect not only the type of substitution of the double bond but also the poiarities of the barbiturates. Decantation of entire solvent phases is used in the various permanganate oxidation procedures. Though a nonquantitative procedure, high precision is not required. An uncertainty of less than 5% is introduced, and time and sample are conserved. The hot alkaline hydrolysis procedure is like that of Broughton ( I ) , but as is desired here, 1N sodium hydroxide gives a somewhat faster rate of hydrolysis. The procedure used does not require addition of water to volume after heating, since an insignificant proportion of the mater is lost. Another simplification is the use of the .A2,, in 1N sodium hydroxide rather than the A A 2 6 , . Greater precision is possible with the latter, but high precision is not required by the system, and a difficulty emountered on addition of ethylenediamine 2HC1 solution to the heated solution is the formation of silicic acid suspension. This results from alkali attack on glass. and the solution must he centrifuged before
reading. Rates of hydrolysis of various barbiturates a t room temperature in strong alkali have been folind useful, but the rates of only the polar barbiturates are rapid enough for convenience. Resistant bsrbiturates are destroyed at rates of less than 0.1% per hour, Rates of hydrolysis given are initial rates whicah apply only over a range of 70 to 100% remaining. If a lower per cent remains a t the time of observation, rates can be calculated from the integrated form of the firstorder rate equation. The partition procedure docs not give as complete physical separation of barbiturates as paper chromatography, hut a t least as good preliminary identification is gi1 en hy the Ratio ValU€? as by Rivalues from paper chromatography. The high rcproducibilitp obtained is to be e from a liquid-liquid system equi under standard conditions. The permanganate and alkali hydrolysis techniques further characterize the harhiturates as members of certain structural classes and allow positive identification. Though chromatographic or countercurrent techniques n oulti be desirable for very complex mixtures, samples containing one to four or posqibly more barbiturates, depending on their natures, are readily analyzed using the ~ y s t e m . Though it has not yet heen applied to nonbiological barbiturate analysi~, the system should
prove useful in identification of pharmaceutical preparations. ACKNOWLEDGMENT
The author thanks Frank McKee, director of the Clinical Laboratories, UCLA Xedical Center, for supplying blood samples, and Raymond Abernethy, head tosicologist, Im Angeles County Coroner's Office, for his kind cooperation. LITERATURE CITED
(1) Broughton, P. M. G., Uzoc7m2. J . 63, 207 (1956). (2) Curry, A . S.,l c t u Pharmacol. Toxicol 13,357 (1957). (3) Curry, A. R., J. Pharm. and Pharmacd. 12 328 (1960). (4) A . S., .Vature 183, 1052 (1959). ( 5 ) Goldbaum, L. R., AXAL. CHEM.24, 1604 (1952). (6) Plaa, G . L., Hall, F. B., Hine, C. H., J . Forensic Sci. 3, 201 (1958). ( 7 ) Stevenson, G. W., ANAL. CHE5i. 32, 1522 (1960). (8) Ibid.,33, 1374 (1961). (9) Stevenson, G.W., University of California. Los Andes. Calif., un-
curry,
published'data.
'
(10) Wickstrom, A., Salvesen. R., J . Pharm. and Pharmacol. 4, '38 (1952).
REcEIvEn for review M a v 5, 1961. Accepted hugust 17, 1961. Investigation supported by research grant B-1106 from the Sational Institute of' Seurological Diseases and Blindnezs of the National Institutes of Health, U. S. Public Health Service. California Association of Criminalists, Los Angeles, Calif ., April 1958. American Academy ,of Forensic Sciences, Chicago, Ill,>February 1959.
Simple Procedure or Conversion of Oxygen of Orthophosphate or Water to Carbon Dioxi termination P. D. BOYER, D. J. GRAVES,
C. H.
SUELTER, and M. E. DEMPSEY
Departmenf of Physiological Chemisfry, University of Minnesota, Minneapolis 14, Minn.
b A convenient procedure for determination of the OI5 content of KHZPO, or H2O is based on heating the samples with guanidine hydrochloride. Oxygen of water and up to two oxygens per K H 2 P 0 4 are converted to COa. The COZ, after removal of ammonia by an H2S04 trap, is used for mass spectrometer analysis. of convt,rting 0 1 8 of orthophosphate to a gas suitable for mass spectrometer analyses include formation of H20 from KHzP4 and equilibration of the H20 with COZ ( 2 , 3, 6. T), heating of 13a3(P04)2 with C ETHODS
1906
e
ANALYTICAL CHEMISTRY
a t 1350' to give CO (d), conwrsion of oxygen of KHzP04to COz by hwting nith Hg(CX)Z (8),and dircct heating of Ag3POd at 1000° to give O2 ( 1 ) . H20 ~amplesarc usually onalyac~dby cquilibration nith C 0 2 (4, 6, 7 ) . COS is the gas usually preferred for m m s spectromrtric d(+xniination of 0l8. JIcthods used for equilibration of water pvr w , or that derived from KHLPO~. nith CO, suffer bpcause of dilution of the 0'8 to be measured, necesity of accurate knowledge of size of water and CO, samples used, 2nd appreciable time required for analyses. In addition, in our eupcrience, equilibration of gaseous H20 and COz hy the hot Pt wire method ( 2 ,6) has
not becn consistently cmipli'tc xith diff ermt equil ihration chambers. This disadvantage appears to bc owrcomc in rquilibration avc~rl(~ratcdh y c,lrctrir discharge. ( 7 ) . Th(x hcating of KH2P04 with Hg(CX)? (8)> although giving COr without dilution, do,^ not rtiatlily >+Id reproducible results ( I ) . The procpilurw ~ i m r i b o dh(Jrc,in ws u l t d from 3, search for rcmtions hy which orthophosphate o x y g i w vould he conveniently convertrd to csrhon dioxide in good yield and without dilut'ion. APPARATUS A N D REAGENTS
In addition to a conventional vacuum train. gas-c~ollwting, anti in:m ~ p c c -
