THE BARBITURATES IN FORENSIC CHEMISTRY DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy- in the Graduate School of The Ohio State University EQr GWENDOLYN BERTHA CARSON, B« S., M* A* The Ohio State University 195U Approved by* Department of Physiological Chemistry ___________ i TABLE OP CONTENTS page A« Introduction 1 B» Historical 1. The Barbiturates ---------------- 2 2* Barbiturate Poisoning - - -------------------- U a) Deaths due to Barbiturates in Pour Counties and the United States - - ------ ----- 11 b) Doses of Barbiturates in Grains - - -- - - 16 3* Barbiturate Regulations------- - ------------ 21 C. Isolation of the Barbiturates ----- --- - - --- - 2h D» Qualitative Tests for Barbiturates ----- — - ---- 26 E. Quantitative Methods for Barbiturates -------- 28 F* Examination of Tissues --- — 30 1# Degradation Products of Barbiturates ------ 3 1 2a Percent Recovery of Unchanged Barbiturates - - - 3^ G. Experimental Procedures --------------- 35 1* Effect of 5$ Potassium Hydroxide on Barbiturates- 36 2 . Effect of 20$ Acetic Acid on Barbiturates - - - - 38 3. Effect of Water on Salts of Barbituric Acids - - 38 k» Barbiturates Extracted from Liver with 5$ Potassium Hydroxide - — ___ ip. 5. Barbiturates Extracted from Liver with 20$ Acetic A c i d ------------------------------- Hi 6 . Barbiturates Extracted from Liver at pH 1 0 HU 7. Refractive Indices of Barbiturates - ------- H6 8 . Barbiturate-Cyanide Reaction ---------------- 50 9» Effect of Atmospheric Conditions on Barbiturates- £2 H* Discussion --- 53 I* Summary ----- - — — ---- — ------------ 63 J. Conclusion — --------- — ------- 6 H K. Bibliography----------------------------------- 6 6 Iw Autobiography--------------- 73 i i LIST OF TABLES page Table I Formulae of Barbituric Acids - ----- 6 Table II Names and Synonyms of Barbituric Acids - - - - 9 Table III Patent Data for Barbituric Acids - - ---- 10 Table IV Deaths due to Barbiturates in New York City - - - - 1 1 Table V Deaths due to Barbiturates in Franklin County, Ohio 12 Table VI Deaths due to Barbiturates in Cuyahoga County, Ohio 13 Table VII Deaths due to Barbiturates in Los Angeles County, California - -- -- -- -- -- -- -- -- -- - lit Table VIII Deaths due to Barbiturates in 1+8 States -------- 15 Table IX Doses of Barbituric Acid Derivatives in Grains - - 16 Table X Minimum Quantity of Barbiturate found in Tissues in Poison Cases ------------------- 20 Table XI Degradation Products Reported for some Barbiturates 31 Table XII Percent Recovery of Unchanged Barbiturates from Urine 3 k Table XIII Melting Points of Barbituric Acids Recovered after 2 k Hour Treatment with 5$ Potassium Hydroxide - - - 37 Table XIV Melting Points of Barbituric Acids Recovered after 2 k Hour Treatment with 20% Acetic Acid - 39 Table XV Melting Points of Barbituric Acids Recovered from their Salts after 2 k Hour Treatment with Distilled W a t e r --- UO Table XVI Melting Points of Barbituric Acids Recovered from Barbiturate-Liver Mixtures after 2 k Hour Treatment with 5$ Potassium Hydroxide - ----- k2 Table XVII Melting Points of Barbituric Acids Recovered from Barbiturate-Liver Mixtures after 2 k Hour Treatment with 2C$ Acetic A c i d ---------- - 1+3 Table XVIII Melting Points of Barbituric Acids Recovered from Barbiturate-Liver Mixtures after 2i+ Hour Treatment at pH 10 - - - -------- 1+5 ill LIST OF TABLES (continued) page Table XTX Melting Points of Pure Barbituric Acids ----- I46 Table XX(a) Refractive Indices of Barbituric Acids - --- U7 Table XX(b) Refractive Indices of Barbituric Acids - - --- H8 Table XX(c) Temperatures at which Refractive Indices were Measured --- k9 Table XXI Melting Points of Materials Recovered from the Barbituric Acid-Cyanide Reaction Mixtures - - - - $1 Table XXII Melting Points of Barbituric Acids after Exposure to Atmospheric Conditions at Room Temperature - - $2 i v ACKNOWLEDGEMENT The author wishes to acknowledge the assistance of Dr* Clayton S* Sbiith, her preceptor and Chairman of the Department of Physiological Chemistry and the helpful suggestions of Dr* Helen L* Wikoff, Associate Professor of Physiological Chemistry, in carrying out this investigation She also wishes to acknowledge receipt of statistical data on deaths due to barbiturates from the Coroner's Office in New York City; Cuyahoga County, Ohio and Los Angeles County, California. The author also wishes to acknowledge gifts of the following barbituric acid derivatives used in making the determinations investi­ gated in this dissertation; Allyl-n-butyl barbituric acid; Butethal; Ethyl allyl barbituric acid; Mosidal; Pentobarbital; Phenobarbital and Pentothal from Abbott Laboratories. Nostal; Pemoston and Sigmodal from Ames Company, Inc* Dial from Ciba Pharmaceuticals, Inc* Alurate from Hofftaan-La Roche, Inc* Amobarbital and Secobarbital from Eli Lilly and Company* Rutonal from May and Baker, Ltd., Dagenham, England* Butisol from McNeil Laboratories, Inc* Ortal from Parke, Davis and Company, Sandoptal from Sandoz Pharmaceuticals* Delvinal froip Sharp and Dohme, Inc* Ipral from E. R. Squibb and Sons and Cyclobarbital; Evipal and Mephobarbital from Winthrop-Steams, Inc* THE BARBITUATES IN FORENSIC CHEM ISTRY A.’ INTRODUCTION Forensic chemistry may he defined as chemistry applied to the solution of legal problems which may arise in connection with the administration of justice* This field covers the application of chemistry to both criminal and civil investigations*1 The forensic chemist may be called upon to examine blood stains; clothing; counterfeit coins; documents; dusts; various types of fibers; strings and ropes; hairs and textiles, in addition to making chemical analyses of drugs, medicines and vital organs# Of the numerous drugs and medicines which might be involved in forensic chemistry, the sedatives are by far the most important* The most widely used present day sedatives are the barbiturates# With the advent of the barbiturates, the older hypnotics such as chloroform, chloral hydrate, paraldehydes and urethanes have only a limited use«' A study of accidental and suicidal deaths occurring in Los Angeles County during the fiscal year 19£3-195>U reveals more then 2h% increase in deaths due to barbiturates since 19k9 -1 9 $ 0 * It is obvious that with barbiturates involved in so many medicolegal cases, accurate methods for th9 detection of these compounds in tissue, blood and urine should be developed*1 While some of the barbiturates are readily detected by the older methods, others defy detection due principally to the fact that they are metabolized in the body and methods for the detection of the metabolites have not yet been developed in all cases#4 This being the fact, the present investigation was undertaken to -2- iraprove old methods or to develop new ones for the detection of the various barbiturates* B. HISTORICAL 1* The Barbiturates Malonylurea, prepared by Conrad and Gutzeit in 1882, was supposedly naned after a 'Miss Barbara".* Other theories as to the name are (1) that Baeyer called the compound barbituric acid because he considered it to be a "key" to a series of such compounds and (2 ) that the name was selected because the compound was prepared on a St* Barbara's Feast Day * Barbituric acid, which is a combination of urea and malonic acid, forms the basic structure for this series of compounds* The two hydrogen atoms at position $ in barbituric acid are quite reactive and may be replaced by various radicals (R and R*). The bar­ biturates may be named according to the substituent groups in position 5. _________ HYPNOPHORE GROUP HN - C = 0 H !HN -_C « 0 B? ^ \ / S ~ X / HO - Cx ^ C \ HO - Cx C ’ N - C - 0 H - C = 0 i_ JRJ, Barbituric Acid Disubstituted Barbituric Acid The first disubstituted barbituric acid derivative to be prepared was the diethyl compound made by Snil Fischer and J* von Mering in 1903 and later patented under the name "VERONAL"* The compound was VERONAL from the Latin word "vera" because Fischer believed it to be 2 3 the true hypnotic and introduced it as a hypnotic In 1903 • This compound has been officially known as BARBITAL since 1926 * Further investigations established the fact that substitution of dissimilar hydrocarbon groups on the side chain produced compounds which had clinical advantages over BARBITAL*1 The next barbiturate to appear was phenylettyl barbituric acid, prepared by Horlein in 1911* This compound, also known as PHENOBARBITAL, was introduced therapeutically several years after BARBITAL Tinder the trade name of WLtMINALH but became official at the same time as barbital in 19264** HN - C - 0 C,HS / \ / HO - C n N - C^* O f ij PHENOBARBITAL 6 In 191o , the parent compound was modified by having one of the radicals, an ethyl group and the other a methyl butyl group*1 This product known as PENTOBARBITAL or NMBUTAL was developed by the Abbott Laboratories*' AMYTAL developed by the Eli Lilly Company in 1921 was shovm in 1930 6 v by Shonle, et al and Volwiler, et al to be an isomer of PENTOBARBITAL•' These compounds had the same empirical formula but the methyl group in the alkyl side chain of AMYTAL was linked to the/carbon atom whereas, the methyl group was attached to the a carbon in PENTOBARBITAL^ HN - C » 0 C2HB HN • C - 0 C2HB / 2 s ^ \ / 36 - HO ~ C' X / \ (a) w \ / V«>, s N - C^" 0 CH2«CH2-CH"CH3CH2«CH2**CH"CH3 'N -• C^-C'« 0 c h -(c2) h 2)2-c h 3 I t c h 3 c h 3 AMYTAL PENTOBARBITAL In later years several other barbiturates were synthesized and marketed for clinical use* Table I shows some of these compounds,