Identification of Malonyl Urea Derivatives I f rared Absorption in Toxicological Analysis C. J . UMBERGER AND GRACE ADAMS Chief Medical Examiner's Ofice, New York, N. Y. Because barbiturates are so frequently encountered in toxicological exaniinations, a reliable procedure for their identification is essential. Chemical methods are not available for differentiating between the barbiturates. In a study of the infrared absorption spectra of these compounds, the spectra of the purified compounds, run at optimum concentrations in chloroform solution, showed significant deviations in the minor absorption bands. The variations permitted a classification and a method of identification of the different members of the series based on their infrared ahsorption characteristics.
B
-1RBITURATES are the organic poisons most frequently encountered in the toxicological examination of organs and body fluids from cases aut.opsied in the Medical Examiner's Office. They are readily detected in tissue extracts by t,he sen.+ tivc Koppanyi reaction (S), although related sedatives such as Sedormid, Carbromal, and Broniural, which are frequently used in combination wit'h the barbiturates, do not give this reaction. -4 riegativr Koppanyi reaction is proof of the absence of barhiturates. However, further invest'igation of the compound giving n positive reaction is necessary for identification, as the test is not specific. The minimal lethal dose for this class of drugs varie.9 from low values for the short-acting nicmbers, such as Pentothal a n d the @-bromoallylderivatives, to relatively high concentrations for the longer acting Luminal and Barbital ( 2 ) . Therefore, although thrre is proof of the presence of a nialonyl urea derivat,ive, a quantitative determination of the concentration is not sufficient evidence to establish barbiturate poisoning as the cause of dt,ath, except in unusual cases. Because it t'akes a different amount, of each barbiturate to produce death, the specific barhiturat,e present, and its concentration in the t,issue, must be known. Individual members of this clam of sedatives may he idrntified by the optical properties of the crystals and derivatives and by thcir micro melting points ( 4 ) . The procedure, howevrr, is laliorious and special methods are required t o obtain crystals in a state of purity high enough for the reproducihility of crystal hahit on which the identification depends. The growing tendency of pharniaceutical houses t o market mixtures of barbiturates of varying activities has greatly increased the difficult,ies of purification for chemical identification. I n the toxicological analysis of tisruc extracts containing organic compounds, the infrared spectral curve is often t h e ollly procedure by which cert'ain of the chemically nonreactive drugs m:Ly be identified with certainty. The sensitivity for detect,ion by infrared is considerably lc~ssthan in ot,her analytical procedures. Concentrations varying betwen 10 and 30 mg. per ml. of solvent are usually required and thc terhnique is limited, therefore, to those drugs whirh have sufficiently high minimal lethal closes to allow adequate recovery from tissues. I n spite of the rather high concentration limit, the limit of identification can be made adequate for many drugs by using small volumes of solvent. The infrared cell with a 0.1-mm. cell depth requires only 0.2 t o 0.3 nil. of solvent and, by using microtechnique in preparing the solution, absorption curves with more than adequate extinction can be obtained with 2 t o 5 mg. of sample. The relatively IOTV spectral sensitivity has a particular advantage in toxicological analysis. The normally occurring extractives from brain tissue do not show a significant absorption and comparatively clear-cut absorption curves are obtainable without the necessity for special purificatoin. Sormal liver extracte do shorv a measurable infra-
red absorption and, for this reason, the brain is a much better or gan for the analysis. Barbiturates and related compounds show infrartd absorption characteristic for the malonyl urea structure and, iri acute poisonings, recovery of the compounds from the organs is adequate for the analysis. Their chemical structures are closely ~,cxlated,so t.hat the general absorption pattern is typical for the group. This spectral pattern, therefore, proves t h a t the positive Koppanyi reaction was given by a malonyl urea derivative and not, some other compound. I n spite of the similarity in infrared absorption of a number of the members, the minor differences in t h e spectra are sufficient to permit identification of individual compounds when absorption data are utilized in conjunction with micro melting points. Barbiturates are isolated from tissue and body fluids in the routine extraction for organic poisons ( 5 ) . Chemically, they act as phenols and appear in this fraction in the solvent eltraction procedure for tissue extracts (6). Vacuum sublimation of the residues from the solvent extraction will free t,he iaolat,ed compounds from all but negligible amounts of t,he normal tissue extractives ( 1 ) . I n applying infrared t o the toxicological analysis of an unknown compound, it is not practical to adjust the concentration by diesolving a weighed sample in the solvent,. Only micro amounts of the drug are isolated from the tissue, and the recovery in different cases is very variable. The absorbing ability of individual eompounds varies over a wide range and, in mixtures, the minor component may be overlooked at lower dilutions. However, controlled concentration is essential for the identification of individual mcmbcrs of a series, since the ratios of the ext,inctions a t two wave lengths may vary appreciably with the changes in concentration i n the solvent. In order t o meet the requirements of controlled concentration with variable sample weight and variable absorbing ability, i t is necessary t o run a series of absorption curves on t h e unknown, darting with a concentrated solution of the drug and then diluting down in steps until optimum extinction is obtained.
1309
PROCEDURE FOR I h F R A R E D A b 4 L Y S I S
Chloroform is used as the solvent. The sample contained in a microbeaker is dried by remaining overnight in a desiccator. Then 0.4 ml. of chloroform is added and mixed with a micro stirring rod. The solution is drawn into a 1-ml. syringe and is then placed in the liquid cell with rock salt windows of 0.1-mm. path ]en th. .4fter the absorption curve of this concentrated solution f a s been run, the solution is withdrawn from the cell with the syringe and an equal volume of solvent is d r a n n into the syringe. Its contents are mixed by rotating the syringe, the absorbing cell is recharged with the approximately 1 to 1 dilution, and a second curve is made on the same paper, using a different colored ink. The dilution process is continued until a suitable curve is obtained. Usually, three trials are adequate
A N A L Y T I C A L CHEMISTRY
1310 Table 1. ~~
A1
Compound Amytal Butisol
...
2.93.0 2.93
,..
2.94
2.83
3.023.07
... .
3.3 3.3
3.22
3.3
-
3.353.4 3.35
3.453.52 3 , 4.5
3.35
3 15
VW
3.1
...
2.94
.
Xeonal
.,,
2.05
...
,
3.1
3.22
3.3
3.1
3.22
3.3
.; 8
.j 77
5.8 5 85
5.77
5 84
3.33
3.45
5.77
5.84
3.33
3.45
5.78
5.84
2.94
, . .
3.1
3.22
3.3
3.47
3.4
..
2 9.5
3.1
...
3.3
3.4
3.48
2.93
3 1
3.21
3.3
3.38
3.47
Sandoptal
...
2.95
3,1
3.23
3.3
3.37
3.48
...
W
VW
3.12
3.24
5.8
5.9
5.75
5.83
vw
5.78
5.83 vw 5,87
vw
VW
3.3
3.38
3.48
5.78
Cyclopal
2.95
Delvinal
2 95
Luminal
B2
... ,..
2.97
3.1
3.2.5
3.3 VW
VU.
3.1
3.23 vw 3.25
3.3 vw
3.38
3.1
3.4
...
5 78
5.85
6.1
W
5.m
5.75
5.85
W
W
3.37
3.52
5.75
V ,v
W
2.94
..
3.1
3.22
3.3
...
3.1
3.22
3.3
3.33 w 3.37
3.47
2.85
vW
VW
N
W
W
2,9
