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 - C x 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"CH3 'N -• C'«C^- 0 c h -(c h 2)CH2«CH2**CH"CH3 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, their trade names and structural formulae* Table II lists names and synonyms

of barbituric acids.

Table III based on the dates when the patents were issued shows the approximate time of the commercial appearance of the individual barbiturates* 2* Barbiturate Poisoning*'

Poisoning both suicidal and homocidal has undergone many stages from

Socrates and his hemlock through the Borgias with their arsenic in the

Middle Ages to bichloride of mercury, phenol and cresols (lysol) of recent years* At present, the barbiturates far outnumber other preparations for the sinister purpose of suicide* The significance of this last statement is clearly shown in tables IV to VIII* Tables DC and X show the wide variation in therapeutic, toxic and fatal doses of barbiturates*1

It should be apparent from the data presented that there should be some regulation of the traffic in barbiturates*

In 192*>, Leake and Ware discussed 6l cases of BARBITAL poisoning which they observed in a Los Angeles Hospital* Nineteen (19) of the cases were of suicidal intent* At that time there was no restriction on the sales of the drug in California^ however, since 1931* the sale of barbi­ turic acid derivatives has been forbidden in California except on a physician*s prescription*1 9 Achard in 1929 reported his observations on the extensive use of barbituric acid derivatives and the great increase in cases of voluntary poisonings* 10 Haubrich in 1934* advocated the restriction of the indiscriminate sale and use of the barbituric acid preparations because they were habit forming^ He also recommended that preparations which were available only on a physician* s prescription not be issued more than once on the original prescription# IX In 1939, Tatum discussed the barbiturate problem primarily from the pharmacologist* s point of view, listing substances which were synergistic with or potentiated the action of this group of drugs. Table I FORMULAE OF BARBITURIC ACIDS abtrt Drvtv Drvtv Drvtv Derivative Derivative Derivative Derivative Barbiturate

vCHg-CB«CH2 ,CH^“CHb CH2 ,c h 2- c h «c h 2 ^ C H 2-GH»CH2 .c h 2-c h -c h 2 R R R R R ^ CHgf-CHg CH^-CH-CHa n CH ( C%)2 n CH2-CH2-CH2-CH3 ^ c h 2-c h (c h 3)2

Ethyl aUjrl Dial Aprobarbital Allyl^-butyl Allyl barbital barbituric acid barbituric acid

NLGE _ isomers Siilfur Bromine ANALOGUES Bromine N-methyl c h 2-c h »c h 2 R‘ S CH (CH3)2 Narconuraal

,CH2-GBr-CH2 R N C H (c h 3 )2

Propallylonal

CH2-CBr“CH2 N-methyl R* ^CH (CH3)2

Eunarcorx

R *• barbituric acid radical ^CH2-CBr-CH2 R*« N^nethyl barbituric acid S*R X CH (CH3)2 radical Thionareon Table I (Continued) FORMULAE OF BARBITURIC ACIDS 8 6 O J in « ? p K ? / ° Y m Derivative Derivative Barbiturate Y « 8 « Y « N./ « M M to ' \ O 04 P4 h 9 M - A e i O 0? V ft) 1 rmn Sulfur Bromine Ci / \ ANALOGUES O h B g V •33 a? Y a? * O (4 ______A ■a SI SI o ixj V W V Y w « / \ Y tu tfl s ✓ \ ri t O « M N « cr I t fri V j?

Surital Kemithal Table I (Continued) FORMULAE OF BARBITURIC ACIDS w /'CgHs / C2H5 /GgHg /CgHs vCgHg R R R R R H*I ' 0 * 'c h -c h 2- c h 3 ^ c h 2-c h 2-c h 2-c h 3 n c h -c h 2- c h 2-c h 3 ^ c h 2- c h 2-c h (c h 3)2 d* t I g h 3 c h 3 o% Barbital Butabarbital Butethal Pentobarbital iscsners Lsome:

R* 13.1 N>C2H6 COMPOUNDS MISCELLANEOUS ANALOGUES H*II H Metharbital <3

S'S? S-R S-R CH-CH2-CH3-CH3 ^ CH2-CH2-CH (c h 3)2 t d CH, £ Thiopental Thioethaayl isSSJers /CgHg / CH2-CH2-CH3 / c 2h 5 CeHg R R R S-R' C»CH-CH2-CH3 'c h 2-c h 2-c h 3 CH (CH3)2 c h 2-(c h 2)4 c h 3 n c h 2-c - c h 3 V CH,3 c h 3 Vinbarbital Proponal Probarbital Hexethal Methallatal

.c h 3 -CH, ^CjjHg ^ C 2H5 R*^« R. R»,:0 Q R« O Rutonal'O Hexobarbital Phenobarbital ProminaL Cyclobarbital Table II ______NAMES AMD SYNONYMS OF THE BARB IT URIC ACIDS STUDIED______

ALLYLBARBITAL - allylbarbituric acid; sandoptal ALLYL-n-BUTYL BARBITURIC ACID - idobutal AMYTAL - amobarbital; amylobarbitone; 5-ethyl-5-isoaroyl barbituric acid APROBARBITAL - ^-allyl-^-isopropyl barbituric acid; alurate; isonal BARBITAL - barbitone; deba; diethyl barbituric acid; doraonal; hypogene; malonalj xnedinalj sedeval; uronal; veronal; yesperal 5-(2-BRCMAXLIL) -5-(1-METHYLBUTYL) BARBITURIC ACID - p-bromallyl sec-amyl barbituric acid; rectidon; sigmodal BUTABARBITAL - butabarbitone; butisol; 5-ethyl-5-methylpropyl barbituric acid; 5 -ethyl-5 -secbutyl barbituric acid BUTALLYLONAL - p-bromallyl-secbutyl barbituric acid; 5-(2-bromallyl)-5- secbutyl barbituric acid; pernoeton; pernoston BUTETHAL - 5-butyl-5-sthyl barbituric acid; etoval; neonal; saneiyl CYCLOBARBITAL - cyclobarbitone; ethyl cyclohexenyl barbituric acid; namuron; palinum; phanodoni; phanodom; tetrahydrophenobarbital DIAL - diallyl barbituric acid; allobarbital; allobarbitone; allyl allyl barbituric acid; curral; 5j5-diallyl barbituric acid ETHYL ALLYL BARBITURIC ACID - dcwmin HEXETHAL - 5-ethyl-5-hexyl barbituric acid; hebaral; ortal HEXOBARBITAL - cyclonal; (cyclohexen-l-yl)-l,5-dimethyl barbituric acid; dorico; evipal; evipan; hexanostab; methenexyl; N-methyl cyclohexenyl methyl barbituric acid METHALLATAL - 5-ethyl-5-JJiethylallyl-2-thiobarbituric acid; mosidal PENTOBARBITAL - embutal; 5-9thyl-5-methylbutyl barbituric acid; nembutal; pentobarbitone; pentone; pentyl; 8I4I1 PHENOBARBITAL - barbenyl; barbiphenyl; dorrairal; 5-ethyl-5-phenyl barbi­ turic acid; euneiyl; gardenal; luminal; neurobarbital; nunol; phenobarbitone; phenonyl; phenylethylmalonylurea; somonal PROBARBITAL - 5-ethyl-5-isopropyl barbituric acid; ipral PROMINAL - mebaral; mephobarbital; N-methylethyl phenyl barbituric acid; phemitone PROPALLYLONAL -5-(2-bromallyl)-5-isopropyl barbituric acid; noctal; nostal RUTONAL - 5-®ethyl-5-phenyl barbituric acid SECONAL — 5-allyl-5-methylbutyl barbituric acid; quinalbarbitone; seco- barbital THIOPENTAL - 5-ethyl-5*

1903 German Ib6>k9& Barbital 1912 German 21*7,952 Phenobarbital 1912 U. S. 1,025,872 Phenobarbital

1912 U. S. 1,01*2,265 Dial

1916 German 293,163 Pentobarbital

1918 U. S. 1,2#,951 Probarbital (2 Ca*3H20)

1923 Uo S* 1,W*U,802 Aprobarbital

1921* U. S. l,5li*,573 Amytal 1926 U. S. 1,576,0114. Probarbital

1926 U* S. 1,609,520 Butethal

1927 u. s* 1,622,129 Propallylonal * u* s. l,62l*,51*6 Hexethal Sodium

1929 C. s. 1,739,662 Butallylonal

# U. S. 1,91*7,91*1* Evipal (Hexobarbital) 1930 German 589,9k7 Cyclopal British 3U9,il55 Cyclopal French 38,680 Cyclopal

1930 German 595,175 Evipal (Hexobarbital) French 753,178 Evipal (Hexobarbital)

193U U. S. 1,9514,1*29 Seconal Sodium

* U. S. 2,090,591* Barbituric Acid

1939 U. S. 2,153,729 Thiopehtal Sodium U* S. 2,153,731 Thiopental Sodium

1939 U. S. 2,153,729 (?) Mosidal # According to the numerical order of the patents, the preparations should be placed in this position* Table IV

ACCIDENTAL AND SUICIDAL DEATHS DDE TO BARBITURATES IN NEW YORK CITY*8

BARBITURATE 19hl 191*2 191*3 19hh 19h6 l?t*7 19i*8 19U9 1950 1951 + /-N + $ 3 3 3 3 si 3 ■P •p H ■P rri •p i—1 c C 3 £ (4 (4 "g $ (4 Is *13 XI Jj 3. $ XI © •o <3 Xl Xf XJ XI XI •H •c •ri XI •H Xl •H T3 •ri ■I •ri • § *•ri XJI •H XII •ri XJ •§ •H •H O *rl O *ri O >H •H O ’H •H O -H •H .3 O •S O -ri n O O •ri o •ri o •H O •ri O •H O •ri O o «J 8 tg 8 % 8 % 8 8 3 8 • § AlLonal - - mm 1 mm - mb 2 - ae- - - •» «• - ALurate & Neonal m mm - mm -- • 1 m Amytal 1 1 - 1 1* 1* 1* 1* 3 1* 6 2 3 1 1 Amytal & Seconal 1* - -- •ft --- - «*• - - 1 6 1 Barbital •» mm - 1 1 2 mm 2 - 1 - - - 1 1 Butisol - m mm- -- m- m- 1 Ipral - - 1 m - mb - •* Nembutal (Pentobarbital) 3 mm 1 13 8 19 6 19 12 22 8 15 6 15 5 Nembutal & Phenobarbital - - «• - - •» - 1 Nembutal & Seconal - •a - ---- 1 Neonal -- 1 - m - - - Neopental ------1 Phanodorn - m - •• •• --- Phenobarbital 1 1* 3 6 5 10 5 7 - 10 6 13 2 13 3 Phenobarbital & Seconal - -- •• ------1 1 — Seconal - -- 5 13 9 8 9 1* 15 5 11 8 16 2 Sodium Alurate - - - 1 «*• - - 1 - 1 --- - 1 Tuinsl «■ ------«•• •• - 1 1 1* 1 Unknown 29 17 27 32 37 31 21 20 76 55 1*3 38 6 0 35 83 31* 76 6 3 0 O Total 38 22 31 32 37 3 1 50 1*2 119 78 —•J 58 113 60 127 56 136 79 Total suicides & accidents/ year 60 i i _68___ 92_ 197 ll*5 173 183 215 186 # figures for 191*5 not yet compiled; + Not broken down into types of barbiturates Table V 33 DEATHS DUE TO BARBITURATES IN FRANKLIN COMFY, OHIO

BARBITURATE 19U8 19k9 1950 1951 1952 1553 195U (through November)

Amytal 3 2 3 1 lU •

Barbital 1 2

Pentobarbital 1 2 1 3 ** t»

Pentothal - 1

Phenobarbital £ 23 8 12 21 2 2

Seconal 1 2 3 5 - -

Unknown 1 J L X Jt _ = _a Total for year 12 37 20 16 U? 2 ll Table VI 1 4 ACCIDENTAL AND SUICIDAL DEATHS DUE TO BARBITURATES IN CUYAHOGA COUNTY, OHIO

SEX 19li3 19k$ 19h6 19k7 2 2 k L 1950 1951 1952 *1 4 ■3 3 3 3 3 $ •p •p H •p -P H H "c *3 ■3 3 S3 S3 « "£ S3 ! -i "d © 3_ §i •rl ■§ a *UI 9 XJ *H O XJ 0 XJ »ri ■§ a •8 a a ■s•H O •H O •ri o •rl •ri O S3 •ri •ri o •ri O O O •ri O z O *ri © •S O a a £ * £ a ^ s 3

Female h 8 it 5 8 2 It It 10 - 7 5 - 5 U 2 2 - 2

Total U 8 6 7 2 8 6 12 U 9 11 £ U; 1 1U 8 1 79 22 3 It \jGH I Total accidents, suicides & un­ determined deaths for the year 12 15 Ut 16 20 20 23 16

14 INCIDENCE OF BARBITURATE POISONING AMISSIONS IN CLEVELAND, OHIO HOSPITALS

19U2 19U3 19UU 19U5 19U6 19lt7 19U8 19h9 1950 Total(# of total poisonings)

77 75 87 9 k 129 161 136 123 112 99k 36ol Table VII 1 5 ACCIDENTAL AND SUICIDAL DEATHS DUE TO BARBITURATES, IN LOS ANGELES COUNTYa CALIFORNIA FOR FISCAL YEARS

1910.- 191*2- 191*3- 191*1*- 1915- 191*6- 191*7- 19U8- 191*9- 1950- 1951- 1952- 1953 BARBITURATE 19l|ii 191*6 191*7 191*8 191*9 1950 22SL. 222L. 1951)

Allonal 1 mm- mm- • * 1 1 1 2 1 (Alurate) (Aprobarbital)

Amytal - 3 3 8 - 1 6 8 5 3 - 5 3 (Amobarbital)

Barbital 1* mm 1 12 5 11* 1 1 1 - - - - (Veronal)

Barbiturate 13 22 20 37 61* 78 66 70 1*9 88 103 123 Ill (Unclassified)

Delvinal «• - 1 - - m m - - - - 1 - -

Dial 1 m m

Ipral - - - - - m m m m 1 - 1 m m - -

Luminal 8 2 16 10 21* 23 31 27 16 20 10 11 17 (Phenobarbital)

Mebaral - m m - - mm «•> - 1 - 2 --

Nembutal 1* 5 6 19 8 20 15 1*1 39 1*0 26 25 ; 39 ( Pentobarbital)

Secobarbital m 9 1 10 12 18 19 26 28 19 16 19 13 (Seconal) Tuinal -- 1 1 2 2 _ 5 3 JL TOTAL 31 1*1 U8 96 113 151* 139 175 ll*2 171* 161* 188 189 -15- Table VIII Xe SUICIDAL AMD ACCIDENTAL HEATHS DDE TO BARBITURATES IN U8 STATES

STATE 1??6 1221 I22i 1222 19itP !L2ki 1 ^ 2 19it3 19U£ Alabama 3 10 it it 3 2 2 » it •ft Arizona m 2 6 it It 3 3 1 3 «» Arkansas 2 1 1 l 2 3 2 2 1 m California 21 31 29 32 56 63 52 93 1 08 193 Colorado •m 3 2 it 2 7 8 2 it - Connecticut 6 9 9 10 9 8 8 6 10 •ft Delaware 1 -» -- -- •ft- •*- » — Florida 8 10 13 10 12 lit 5 8 10 ■ft Georgia 5 8 3 3 3 6 - it 2 9 Idaho 2 it 2 l 3 1 l — 1 - Illinois 33 63 56 51 70 70 2it 31 33 6o Indiana 11 18 12 16 27 17 12 9 lit - Iowa k 9 15 9 8 lit 5 5 8 9 Kansas 3 h 3 9 11 3 3 3 1 9 Kentucky 1 it 6 5 3 it it 2 3 6 Louisiana it 1 it 9 2 it 5 1 2 • Maine h 3 1 it 3 5 it - 3 • Maryland it 1 1 1 5 3 5 7 7 9 Massachusetts 22 20 20 22 2it 35 26 28 22 it2 Michigan 11 lit 16 26 28 31 13 lit 11 - Minnesota 8 lit 11 9 9 12 12 13 lit 11 Mississippi •* 3 -- — 9 12 lit 6 3 it Missouri 6 13 16 12 32 7 it 10 n m Montana 2 •• - 1 2 1 1 1 it m- Nebraska ft* 2 1 it 6 3 3 2 i - Nevada 3 - 1 2 1 •• 1 1 ■». — New Hampshire 1 1 it 2 1 9 3 1 - - New Jersey 7 it 7 7 17 13 9 12 11 - New Mexico 1 3 1 2 3 it - 3 2 ■» New York 57 h9 62 58 6o 65 73 87 98 m North Carolina it 2 1 1 5 2 it it it - North Dakota 1 - -— 3 1 2 •»< Ohio 28 21 16 32 55 71 it2 38 38 72 Oklahoma 5 6 6 3 l 2 3 it 5 Oregon 5 6 5 it 2 6 5 1 h 10 Pennsylvania 12 15 10 15 18 20 18 21 18 - Ehode Island 1 1 2 l 1 1 1 1 2 South Carolina 3 3 3 3 1 2 - •ft 3 • South Dakota - 1 •a- m- •» 1 1 - Tennessee k 5 2 it 2 2 3 1 5 ■ » Texas 7 6 5 8 12 16 7 13 11 a * Utah 2 6 5 3 1 1 1 — 3 m Vermont m 1 2 - 3 1 3 - 1 Virginia l ■a- - 2 2 3 1 it 7 it 'Washington 16 17 10 5 8 9 7 12 n 15 West Virginia 5 3 5 l 5 7 9 it 7 Wisconsin 7 12 19 lit 15 17 13 9 6 Wyoming - 1 ••- - - 2 - - 1 • Table H DOSES OF BARBITURIC ACID DERIVATIVES IN GRAINS

NAME OF DRUG THERAPEUTIC POSE* TCKIC DOSE* FATAL DOSE*______Oral Intravenous Minimum Median Maximum Minimum Median Maximum Alphenal i.S-517

Alurate 1-217,18,19

Alurate Elixir 6x ounces2 0 _ 17,21-23 33 23 23 30 _,23 20 Amytal 1.5-5 3 25 108 2U 25 io5 18 22 • 22 0.33-12 22.5-30 304*5 24 , 2 5 H 7.5-22.5^ 1*8

, 27 Butisol 0.5-1.5 11*9 Table IX (continued) DOSES OF BARBITURIC ACID DERIVATIVES IN GRAINS

NAME OF DRUG THERAPEUTIC DOSE* TOXIC DOSE* ______FATAL DOSE*______Oral Intravenous Minimum Median Maximum Minimum Median Maximum .17 Qyclopal 0.83-2.5 , 2 1 2-1* -17,21 Delvinal 0.5-5 .17,18,21,22 .22 22 Dial 0.5-5 *■ 9 30-37.5 above 37.5 .24 - , 21 24 24 32 ,20 >7.5 1.5-H 30 72 36 .24 24 H Evipal 1.5-5 7.5-15 t , ,21 ,17 U-6 3-6 « .17 Ipral 0.83-7.5 o i*8 2-1* ,17 Hosidal 2.5 .17,21 27 Neonal 0.83-1.5 * 230 « ✓ 18 0 .83-6.7 _ .17 ,18 Noctal 0.83-5 . 1 7 .1 8 ,2 1 Ortal 3-6.7 ' * Table IX (continued) 1DSES OF BARBITURIC ACID DERIVATIVES IN GRAINS

N M E OF 33RIX? THERAPEUTIC BOSE* TOXIC BOSE*______FATAL POSE*______Oral Intravenous Minimum Median Maximum Minimum Median Maximum . 17*18,21,22,24 23 ,.23 23 23 26 Pentobarbital 0.5-3 12 Ui 15 15 30 24 , ^ 26 5-15 U8.7 , 20 k9

. 27 2Ul 17 17 Pentothal 0.83-1.3 0.83-1.2 18,21 I COH Pernoston 3 9 I .22 „ . 2 2 3-7.5 7.5-15 21 17 33 27 34 Phanodom 1 .5 -3 1 .5 -6 It50 276 300 18,22 22 1.5-6 5 18 17.18 . ,22 20.23 Phenobarbital 0.2-3 * 60-75 25-26 9 21,22,24 22,26 0.5-5 75-i5o Table IX (continued) DOSES OF BARBITURIC ACID DERIVATIVES IN GRAINS

NAME OF DRUG______THERAPEUTIC DOSE*______TOXIC DOSE*______FATAL DOSE*______Oral Intravenous Minimum Median Maximum Minimum Median Maximum

Prominal 0*5-10 21 Rutonal 1-2 .36 3? 1-15 120 1 7 .1 8 .2 1 Sandoptal 3-13 * *

. 17.21 ,38 23 25 Seconal 1*5-3 28.5 15 30 18 26 1.5-5 U8.7 . 2 1 Sigmodal 1*5-3

* The discrepancies in the various doses of barbituric acids illustrate that a definite quantity can not be set as the toxic or fatal dose* A dose is considered toxic when sedation lasts longer than anticipated from 3 9 the quantity of drug administered* Wagner states that the therapeutic dose of the barbiturates can not be definitely standardised and suggests starting with the dose ire commended by the manufacturer and varying the dosage, if necessary, to obtain the desired results* Table X

MIN3MUN QUANTITY OF BARBITURATE FOUND/lOO GRAMS OF TISSUE ERCM POISON CASES.

QUANTITY OF TISSUE ESTIMATED ANALYZED BARBITURATE FOUND QUANTITY FOUND FATAL DOSE* Grams Milligrams Grains

100 Amytal 2 .5 10

100 Dial 1 0 ,0 Uo

100 Evipal (Hexobarbital) 2 .5 10

100 Luminal (Phenobarbital) 1 0 .0 Uo

100 Nembutal (Pentobarbital) U.o 16

100 Neonal (Butethal) U.o 16

100 Proponal 1 0 .0 Uo

100 Ortal (Hexethal) U.o 16 100 Seconal U .o 16

100 Veronal (Barbital) 1 8 .0 72

If the quantity of barbiturate isolated from 100 grams of tissue is multiplied by 21*0 , the total amount of drug in the entire body of a 6 0 kilo person (exclusive of the gastrointestinal tract and urine) may be estimated40* -21-

3# Barbiturate Regulations. 41 In 19U6, Robert A# Fischelis reviewed the status of the barbiturate regulation# A summary of his article follows:

As of October 1, 19 k $ , thirty-two states, Alaska, Puerto Rico and the District of Columbia had lews in force which either directly or in­ directly controlled the distribution of barbiturates or of mixtures with other drugs#

The first law dealing solely with barbiturates was enacted by Califor­ nia in 1929#

Wide variations were found in the provisions of the laws regulating the refilling of prescriptions# Since no reference was made to the renewal of prescriptions in fifteen states, it was assumed that refills were permitted.*' An analysis of the labeling requirements which must be observed by the pharmacist showed a marked lack of uniformity in these requirements#'

This lack of uniformity was attributed to the difference of coverage by the various laws#

Illegal possession was made a violation in Arkansas, California,

Georgia, Michigan, Minnesota, Mississippi, South Carolina and Puerto Rico#1

The conditions Tinder which possession was declared illegal were:

1# Possession other than as authorized by law

2#* If not in the original container in which it was dispensed ty

the pharmacist or physician

3# Unless the label showed the name and address of the prescriber

and the name and address of the dispenser

U# Unless furnished on the prescription of a physician, dentist,

chiropodist or veterinarian and -22-

5* Unless prescribed ty a practitioner possessing a valid U* S*

Narcotic license*

Minnesota described a violation as a gross misdemeanor, while the

District of Columbia, Puerto Rico and twenty-two states designated the violation as a misdemeanor* Violations in other states were not classified* 16 Goldstein in bis review of the barbiturate situation drew the following conclusions in 191*7 :

1*' Deaths caused by barbiturates were increasing especially in

those states with large urban population and in instances where

191*5 figures were available, a sharp increase was indicated in

many states*

2*' The state laws as of October 1, 191*5 affecting the distribution

of barbiturates appeared to have little effect on the increase

in mortalities due to barbiturates*^

3* The ratio of barbiturate poisonings to the total number of cases

admitted to the same hospitals for the periods of 1 9 2 8 - 1 9 3 7 and

191*0*191*5 showed an increase of 8 6 $ in the frequency of occur­

rence of barbiturate poisonings in the 191*0 -191*5 period*

1**‘ The ratio of barbiturate poisonings to all types of drug poison­

ings, with the exception of carbon monoxide and alcohol, showed

a 1 9 3 $ increase in the frequency of barbiturate poisonings in

the 191*0-191*5 period over the 1928-1937 period for the same

hospitals*

5*' A total of 1,01*9,785 cases were admitted as poisonings during

the period from 1928-1937* One-seventh (l/7) of these cases

was due to barbiturates*1 During the 191*0-191*5 period a total

of 1,060,275 cases were admitted as poisonings* One-fifth (l/5) -23- of these cases was due to barbiturates* In other words, the

barbiturate poisonings increased from lk»3% to 20% from the

1928-193^ period to the 19UO-19U5 period for the same

hospitals*

6 , United States statistics indicated that fatal poisonings by all solid and liquid poisons were decreasing while the yearly

fatal barbiturate poisonings were increasing*

This review showed that suicides from barbiturates were still increasing in 19U7 and as a result a corrective uniform state legis­

lation was stressed*

In 19U7 an attempt was made by the Ohio State Pharmaceutical Association to secure the enactment of a barbiturate law; however, the

bill failed to be enacted* Another attempt was made in the 19li9-1950 session of the legislature and with the cooperation of the Ohio State Medical Association, this bill was passed by the legislature and signed by the Governor on May 12, 19k9» The law which became effective in

August, 19h9 was modelled after the Uniform Barbiturate Control as suggested by the American Pharmaceutical Association and is known as the Uniform Barbiturate Act of 19i+9« The act essentially made refills illegal on all existing prescrip­ tions filed prior to August 12, 19h9» Thereafter an original written prescription was required each time that any barbiturate or preparation of barbiturate was dispensed. Prescriptions and all invoices of receipts of barbiturates had to be kept on file for two years and to be available for inspection by authorities at any time0 - 211- ISOLATION OF THE BARBITURATES

Methods for the detection of barbiturates usually involve prelimin­ ary treatment of the tissue with acid or base, with or without removal of the proteins, followed by extraction with an immiscible solvent**

A* (a) Chloroform was the solvent selected by several investiga- 42 tors* Koppanyi used chloroform to extract protein free

filtrates prepared from tissues digested by 5 percent KOH*' 43 Adrian! mentioned the extraction of tissues which had

been frozen, pulverized and acidulated* In the Stas-Otto

procedure, the finely divided tissue acidified with tar­

taric acid is extracted several times with absolute alcohol

which is then evaporated to diyness and the residue dissolved in acidulated water from which the barbiturates

are extracted by chloroform*- The Haines* modification of

the Dragendorf process is similar to the Stas-Otto pro­

cedure and differs principally in the longer time required

because acidulated $0 percent alcohol followed by increas­

ing concentrations of alcohol is used in place of the

initial treatment with absolute alcohol*

(b) Several investigators used ether for extracting barbitur- 44 ates* Valov digested his specimens with alkali before preparing the protein free extracts for ether treatment** 46 Kozelka extracted a protein free filtrate prepared from 46 an acid digestion mixture, Klingefuss and Reinert extracted urine with ether to remove Impurities, then ob­

tained the barbiturates from the urine by further ether extraction after acidulation.' Gould and Hine obtained

barbiturates from serum by a continuous ether extraction 48 without previous removal of proteins. Stainer reversed

the order of procedure in separating barbiturates and

salicylates occurring together in tablets. Both were

dissolved in ether from which the salicylates were extract­

ed by aqueous sodium bicarbonate solutions. A chloroform- 49 ether mixture was used by Warren for extracting

barbiturates from a solution prepared from tablets

dissolved in aqueous sodium hydroxide and then acidified

with HCl. so (c) Brundage used petroleum ether to extract barbiturates

from mixtures of aqueous tissue extracts* activated charcoal

and Planter of Parish Preliminary extraction with petroleum 51 ether was used by Pucher to remove impurities from urine

while further extraction of the urine with diethyl ether

after acidulation removed the barbiturate. 62 B. (a) Stolman and Stewart absorbed the barbiturates on synthetic

magnesium silicate from which they were eluted with methyl

alcohol. 63 (b) Franchini and Repetto absorbed the barbiturates on acti­

vated charcoal and then eluted them with alkali.1

(c) Successful paper chromatography of barbiturates in urine 54 has been reported by Algeri and McBay and Algeri and 65 56 50 57 Walker while MohrschulK 9 Brundage and Raventos were

unable to obtain satisfactory results.

Purification of the barbiturates obtained in the above procedures is frequently necessary and several methods have been attempted# Sach 59 and Hanson report the destruction of impurities by oxidation of the crude extract with potassium dichromate and potassium permanganatej

respectively. Other investigators attempted to remove proteins from 60 crude extracts* Fleury and Guinnebault used mercuric sulfate for this 61 62 purposej Zwikker , copper-pyridine# Bachem removed colored impur­ ities from the crude extracts by absorbing them on charcoal# Sublimation

of the crude extract is a successful method of purification according 63 64 65 to Shonle, et al , Herwick and (Settler •

QUALITATIVE TESTS FOR BARBITURATES

Many of the qualitative tests for the detection of barbiturates

include colorimetric procedures# Both the murexide test and Millon's 43 test serve this purpose although Stainer found that MEBARAL and EVTPAL did not give the Millon's test.

Colors produced by the reaction between a cobalt ion and an imide group of the barbituric acid molecule on the addition of alkali have

been extensively used# A pinkish violet or blue color develops when

barbiturates are treated with a solution of cobaltous chloride in

absolute methyl alcohol followed by barium methylate (Zwikker test)# The thiobarbiturates produce a green color with the cobalt reagent# 66 Bodendorf used cobaltous nitrate in anhydrous ethyl alcohol followed by potassium hydroxide in absolute ethyl alcohol and got a blue color 67 as did Kozelka and Tatum who used sodium ethylate in alcohol as the 63 base. Gettler employed cobaltous acetate in absolute methyl alcohol with isopropylamine as the base to produce a violet color while Selwyn -27- 6d and Bark used a more concentrated solution of cobalt acetate and

dilute isopropylamine.

Other color tests for the detection of some of the barbiturates

include Ekkert’s test in which the identification of DIAL, LUMINAL and

PHANODOHN is based on the production of colors following treatment with

formaldehyde and concentrated sulfuric acid. Application of heat to

the mixture produces a fluorescent solution if DIAL is present; a red solution if LUMINAL is present and a reddish-brown solution if EHANODORN 70 is present. Dehusses applied heat to a mixture of m-nitrobenzaldehyde

and concentrated sulfuric acid and got a red color with barbiturates containing the cyelohexenyl radical. Ekkert also used a mixture of

selenous acid and sulfuric acid with which he distinguished BARBITAL

and PHENOBARBITAL by emerald green and wine colors, respectively. 71 Rhodes produced an intense brown color when he heated the phenyl der­

ivative of the barbiturates on a slide with dilute sulfuric acid alone. The odor of ammonia produced when barbiturates are heated alone or with alkali has been used as the basis for an additional qualitative

test. Rhodes71 identified VERONAL, NEONAL, RUTONAL, LUMINAL, DIAL and

APROBARBITAL by crystal formations on a slide when the respective compounds were dissolved in ammonia and acidified with sulfuric acid.

The iron-calcium chloride-iodide reagent of Ludy-Tenger and a

copper-calcium chloride-iodide reagent have also been used to produce crystalline compounds with some of the barbiturates. Colorless to black crystals result from the formation of a copper complex and brown to black crystals from the formation of an iron complex. Kaiser, 72 et al used these reagents and took melting points of the complexes -28-

to identify EVIPAL, LUMINAL, NOSTAL, PHANODORN, PERNOSTON,and VERONAL* 59,73,74 Other investigators identified the barbiturates by deter­

mining the melting point of a derivative that had been prepared 59 74 (frequently the di-p-nitrobenzyl derivative ). 48 Stainer employed various precipitation reactions and the Parra,

test to identify the barbiturates*

QUANTITATIVE METHODS FOR THE DETERMINATION OF BARBITURATES*

Many of the color reactions used in testing qualitatively for

barbiturates have not been satisfactorily adapted for the quantitative 59,75 estimation of barbiturates * The oolors developed by the cobalt 42 reactions fade or are not constant* However, Koppanyi , using cobalt

acetate, anhydrous methyl alcohol and isopropylamine as the base, 76 developed a color which he measured colorimetrically* Krause and Riley varied the reagent concentrations used by Koppanyi and also obtained a 77 color suitable for colorimetric determinations* Bacila and Alcaide ,

using a 5hO filter in an Evelyn photoelectric colorimeter, made a quantitative estimation of' .barbiturates following their reaction with

cobalt nitrate, ammonia and glycerol all dissolved in 96% ethyl alcohol* Unfortunately, the cobalt reaction is not specific for barbiturates but may be produced by any of the following compounds which frequently occur

as impurities* These interferring substances includes acetic acid,

aldehydes, biuret, creatine, creatinine, guanidine, hippuric acid, phospholipids, oxamide, substituted acetamides, substituted acetylureas, 50 theobromine, theophylline and uric acid* Brundage and Gruber found that PENTOBARBITAL and ORTAL yielded metabolic products which were measured as barbiturates by the cobalt test even though these products had no pharmacological action. Spectrophotometric measurements of ultraviolet regions have been employed by several investigators using barbiturates dissolved in 78 47,79,80,81,82 81,83 83,84 acid 5 in alkali 3 in chloroform and in ether • 82 However, one group of investigators reported salicylates and sulfon­ amides as interferring substances. 4 9 Warren weighed the extracted material and determined the melting points. 85 86 Both Bartilucci and Discher and Mattocks and Voshall employed titrations for estimating barbiturates. The former investigators titrated potentiome trie ally with 0.1 N NaOH in an aqueous solution of the barbiturate. The latter pair used a silver, silver-silver chloride electrode system for titrating with silver nitrate* '30'

BMHNATIQBT OF TISSUES

In the examination of tissues one must bear in mind that the drug originally administered may be present unchanged or may be in the form of a metabolite or degradation product.

Therapeutic doses of some barbiturates are believed to be destroyed in the liver. Most canpounds not destroyed in the liver are excreted by the kidneys. The ultra short acting compounds are destroyed in the 87 gut • For convenience the barbiturates are classified as short acting, long acting and intermediate depending upon the length of time of the sedation which they produce.

Short acting compounds: are inactivated chemically by the tissues,

principally the liver. Intermediate acting compounds: are partially destroyed in the body and the unchanged portion excreted by the

kidneys.

Long acting compounds: are eliminated practically unchanged in the

urine.

Table XI showing the degradation products found in tissues after the administration of some of the barbiturates and Table JfJT showing the percent recovery of unchanged barbiturate from urine specimens follow: -31- Table XI DEGRADATION PRODUCTS -BEgflKTED EQiL-SfflB-JARSITBSATES- Drug Administered Compound, Pound in Tissue

( S 8 ) ( M )

CH3 - N - C » 0 H - N - C / V / \ / C sH5 0 * c c 0 - c G ^ \ / N / H - N - C - 0.0 H - N - C » 0 o

PRCMINAL FHENOBARBITAL

(88)(89) CH3 - N - C » O H - N - C » 0 / \ ^ C aHe / \ JJaHs 0 - C C 0 ■ c c \ / x c3h5 V / x c 2h 5 H - N - C - 0 H - N - C - 0

METHARBITAL BARBITAL

(88) CH3 - N - B - 0 H - N - 3 - 0 / V / C aHg / 0 - c c 0 - c \ / \ S X CaH5 CH3 - N - C - 0 H - N -

N,N»-DHMETHYLBARBITAL BARBITAL

(89)

CaHs - N - C - 0 H - N - C - 0 / > ^ C aHs / N ^ C aHs 0 » c c 0 » c c N / N CaH6 \ ✓ V C 2 HB H - N - C - 0 H - N - C “ 0 N-ETHILBARBITAL BARBITAL

(?3 )(oo) CH3 - N - C - 0 H - N - C => 0 / \ .CH(CH3)a / \ CH(CH3)a 0 - C C 0 - C C \ / VCHaCH*CHa \ / X CHaCH«CHa H - N - C - 0 H - N - C « 0 NARCGNUMAL ALURATE -32- Table XI (continued) DEGRADATION PRODUCTS REPORTED FOR SCME BARBITURATES

Drug Administered Compound Found in Tissue

(91) H - N - C - 0 H-N-C-0 / \ . CH, / ' ^ c h 3 0 = c c- 0 - c c%, \ / \ / CHa - N - C - 0 H-N-C-0O ' 0

EVTPAL DEALKYLATED CYCLOHEXENQNE METABOLITE (aa)(93)(94)

H - f i - C « 0 H - N « c - 0 / \ / 0 » c c, 0 — C \ ✓' \ H - N - C - &0 H - N - C - 0

PHANODORM CYCLOHEXENONE METABOLITE

(96) H-N-C-0 H - N - C - 0 / \ ^ C 2H5 0 - c c 0 » c c \ / N CH (CH3) CHaCH2CH3 \ / X CH CH2CHQHCH3 H - N - C « 0 H-N-C-0 ^ C H 3 PENTOBARBITAL HYDROXY DERIVATIVE

(ae) H - N - C - 0 H-N-C-0 / \ X C4H9 / \ ^ C 4H9 0 - c c 0 » C c \ / N CH2 CBr-CH2 \ / X CH2 CO c h 3 H - N - C - 0 H-N-C-0

FEENOSTCN (BUTALLYLONAL) ACETONYL DERIVATIVE

(91)(97)(9S) H-N-C-0 H—N-C-0 / \ .CH (CH3)2 / ^ ,CH (CH3)2 0 » C c ' 0 - C \ / n ch2 cb« o h 2 \ / V CHa CO CH3 H - N - C - 0 2 H-N-C-0 NOSTAL (PROPALLYLONAL) ACETGNYL ISOPROPYL DERIVATIVE -33- Table XI (continued) ______DEGRADATION PRODUCTS REPORTED FOR SCME BARBITURATES______

Drug Administered Compound Found in Tissue

(9 9 ) H-N-C-0 H-N-C-0 / \ ^OjHa / \ ^ c h 8c h 3 HS - C C HS - C C r * ^ / V CH (CH3) CH2CH2CH3 W / N CH CH2CH2CH3 N - C = 0 N - C - O

THIOPENTAL CARBOXYLIC ACID DERIVATIVE

The carboxyl group is believed to be in one of these three positions*

Some of the barbituric acid derivatives are not degraded in the tissues* These compounds, of which BARBITAL is an example, are excreted unchanged* Table XII PERCENT RECOVER! OF UNCHANGED BARBITURATES 1 ft fSHR OF BRUIj 0 TISSUE QUANTITY FOUND RECOVERED AHIINISTRATION EXAMINED IN PERCENT REFERENCE

Amytal intravenous urine 0 100 intravenous urine 1*0-50# in 21* hours 101 Aprobarbital intravenous urine 7% 102 oral urine l*,5-2l*# in 3 days 103 Barbital oral urine 8% in 12 hours id* oral urine 50# in 13-35 hours 105 oral urine ll*-2Q# in 2k hours 60, 101*, 106 oral urine 66-7$% in 21* hours 105 oral urine - 36-90$ in 1*8 hours 10l*, 105, 107 oral urine 50-90% in l*-8 days 75, 103, 3d*, , 107*131 4- oral urine 90-95% in 5 days after 1 theobromine therapy 310 ButallyIonal oral urine 5-17* 93 Cyelobarbital oral urine 2.5-7% 93, 91* Dial oral urine 1*0-$<$> in 3-1* hours -165 Hexobarbital urine 30-1*0# 88 Pentobarbital intravenous urine 0 312 oral urine 0 113-115 oral urine 60# in 21* hours 116 Fhenobarbital oral urine 35-75# in 19 hours 105 oral urine 20# in 9-10 days 103 Prominal oral urine 0 89 Rutonal oral urine 25# in 11* days 317 Seconal oral urine 0 313 - 3 $ - EXPERIMENTAL PROCEDURES

The validity of some of the existing qualitative and quantitative

methods for the detection of barbiturates has been questioned by previous

investigators© Extraction procedures for the isolation of barbiturates

from the tissues give rise to two possible sources of error (a) destruc­

tion of some of the barbiturate through contact with the solvent (acids

or alkalies) and (b) failure of the solvent to dissolve all of the bar­

biturate© This situation might arise if the barbiturate were incomplete­

ly soluble in the solvent or if it had been occluded by impurities (such

as precipitated proteins) and thus was not exposed to the action of the

solvent©

Extraction of the crude aqueous solution with immiscible solvents,

particularly ether, might remove other substances which would be weighed

along with the barbiturate. The use of dichramate or permanganate for

the destruction of contaminating substances in the crude barbiturate

extracts may also be questioned, since some of the barbiturates are

unsaturated and may be oxidized by such treatment. Separation of the barbiturates by adsorption on solids (charcoal, Plaster of Paris) has bean criticized on the grounds that later removal of the barbiturates

from the adsorbent may be incomplete©

The several satisfactory methods developed for the extraction and

determination of barbiturates in pure aqueous solutions or medicaments

such as tablets have not yet been satisfactorily extended to the deter­ mination of barbiturates in tissues for two possible reasons, (a) Macro­ methods used in the former instances are unsuited for the detection of the small quantity of drug present in the tissues; (b) No provision has -36- been made for removing contaminants arising from the presence of tissue* Many of the investigators who weighed residues and reported them

as barbiturates failed to ascertain the purity of the substances by

determining the melting points. No identification should be based on a

color test alone since there are other substances besides the barbitur­

ates (particularly proteins) which give the same color tests* However,

because of the limited amounts of barbiturates isolated from tissues, it is seldom possible to prepare chemical derivatives as some investi- 45,59*71,74,118 gators have proposed • In the present investigation we studied many of these points to determine which of the previous methods might be safely employed for extracting barbiturates from tissues* The next step involved the puri­

fication of the barbiturates and the final problem was the identifica­ tion of the material*

The stability of the various barbiturates in water, 20$ acetic acid

and 5$ potassium hydroxide for 2b hour periods was first determined

since these were the reagents most commonly used for extracting tissues*

(a) Determination of the effect of 5$ potassium hydroxide on barbiturates*

Solutions of the 2b barbiturates under investigation were prepared

by dissolving respectively 25 mg. of each In 25 cc. portions of $%

potassium hydroxide. These solutions, each containing 1 mg* of barbi­

turate per cc, alkali stood for 2b hours at room temperature and then were transferred to separatory funnels and acidified with 10$ hydro­

chloric acid, an excess of 2 cc* acid being added to each* Four extractions with chloroform (15 cc. portions) were then made to remove the barbiturates from the acidified solutions. After evaporating the -37-

chloroform from , the combined extracts for each solution, melting points

of the recovered barbiturates were determined and compared with those of the starting materials* The percentage of each barbiturate recovered

was estimated by weighing* These results are included in Table XIII*

Table XIII

MELTING POINTS OF BARBITURATES RECOVERED AFTER 2l+ HOUR TREATMENT WITH 5# POTASSIUM HYDROXIDE Melting Point Melting Point Percent of Recovered of Original Pure Compound______Recovery Material (°C) Barbiturate (°C)

Allylbarbital 62.8 136..5-137 138..5-139 Allyl-n-butyl barbituric acid 1+6.7 •» 123..5-121+ Amytal 50.2 m -11+8.5 1U8 -11+8, Aprobarbital l+o.o 135 -138 li+2 Barbital 8.1+ 182 186 5-( 2-Bromallyl)-5- (1-methyl butyl barbituric acid ioi+.5 157..5 157 Butabarbital 59.8 162 161+ Butallylonal 63.6 131 -132 131 -132 Butethal 21.5 117 -118 123 -121+ Cyclobarbit al 33.3 158 -159 ll+9 -150 Dial 56.0 charred 161+ -165 Ethyl allyl barbituric acid 35.1 155 157 Hexethal 56.6 111 -112 122 -123 Hexobarbital 25.0 charred 139 -ll+O Methallatal 6.8 152 156 -157 Pentobarbital 31. h 121 -122 121 -122 Phenobarbital 17.6 170 -171 171 -172 Probarbital 39.0 198 198 Prominal 1+.8 179 - 172 Propallylonal 31+.3 170 -171 178 -179 Rutonal 8.6 * 215 Seconal 1+5.5 65 81+ -85 Thiopental 77.3 153 153 Vinbarbital 63.8 152 152 -151+

# Material recovered was not crystalline I

-38-

(b) Determination of the effect of 20$ acetic acid on barbiturates*

Solutions of the 2I4. barbiturates under investigation were prepared

by dissolving respectively 2 5 mg* of each in 2 5 cc* portions of 2 0 $

acetic acid* These solutions, each containing 1 mg* of barbiturate per

cc* acid stood for 2U hours at room temperature. The ten (10). compounds

marked * in Table XIV were not completely soluble in 20$ acetic acid.

The preparations were transferred to separatory funnels and 10 cc, of

concentrated hydrochloric acid added to each* Four extractions with

chloroform ( 1 5 cc* portions) were made to remove the barbiturates from

the acidified solutions* After evaporating the chloroform from the

combined extracts of each solution, melting points of the recovered

barbiturates were determined and compared with those of the starting

materials* The percentage of each barbiturate recovered was estimated

by weighing. The results are included In Table XIV*

(c) Determination of the effect of water on salts of barbituric acids*

Solutions of salts of the 2k barbiturates under investigation were

prepared by dissolving respectively 25 mg* of each in 25 cc* portions

of distilled water* These solutions, each containing 1 mg* of barbi­

turate per cc. water stood for 2k hours at room temperature. Ten (10)

cc* of each solution were used to determine the percent of salt present^

the remaining 15 cc* of each were transferred to separatory funnels and

acidified with 1 0 $ hydrochloric acid, an excess of 2 cc* acid being

added* Four extractions with chloroform (15 cc. portions) were then

made to remove the barbiturates from the acidified solutions* After

evaporating the chloroform from the combined extracts for each solution, Table XIV MELTING POIHTS OF BARBITURATES RECOVERED AFTER 2li HOUR TREATMENT WITH ZQ$ ACETIC ACID

Melting Point Melting Point Percent of Recovered of Original Pore Compound______Recovery Material (°C) Barbiturate (°C)

Allylbarbital 73.5 138.5-139 138.5-139 Allyl-n-butyl barbituric acid 55.7 125.5-126 123..5-12U .Amytal 87.0 . 1U8 -1U8.5 1U8 -1U8 . Aprobarbital 70.2 1U2 1 U2 Barbital 72.lt 186 186 *5- (2-Bromal3yl)-5-(1-methyl butyl barbituric acid 100.0 155 -156 157 Butabarbital 70.8 163 16U Butallylonal ioU.i 128 -129 131 -132 Butethal 75.6 123 -12U 123 -121+ Cyclobarbital 7U-7 157 Ht9 -150 *Dial 77.U 163 16U -16$ Ethyl allyl barbituric acid 38.0 157 157 ■aHexethal 75.3 123 122 -123 ■ifHexobarbital 7U.5 1U2 -143 139 -1 J4.0 ■HMethallatal 71.2 155 -156 156 -157 Pentobarbital 80.6 121 -122 121 -122 Phenob arbital 95.6 171 171 -172 *Probarbital 7 8.5 198 198 ttProminal 97.7 172 172 *Pr op al lylonal 92.0 170 -171 178 -179 Rutonal 95.1 215 215 Seconal 86.2 65 Bb -85 ^Thiopental 86.7 153 153 Winbarbital 102.3 152 152 -15U * These compounds were not completely soluble in 20$ acetic acid, there­ fore this method is not applicable for their isolation.

melting points of the recovered barbiturates were determined and compared

with those of the free acids. The percent of salts present at the end of

the 2it hour period and the melting points of the recovered barbituric acids together with those of pure barbituric acids are recorded in

Table XV. V

-Uo-

Table XV

MELTING POINTS OF BARBITURATES RECOVERED FRCM THEIR SALTS AFTER 2h HOUR TREATMENT WITH DISTILLED WATER

Percent Melting Point Melting Point Recovery of Extracted of Barbituric Compound of Salt Acid (°C) Acid (°C)

Allylbarbital Sodium 55 136 -138.5 138.5-■139 Allyl-n-butyl barbituric acid 85 121 -123 123.5--121; Amytal Sodium salt 100 llj.8 -11*9 11*8 -ll*8., Aprobarbital Sodium 76 1U2 -H*3 1U2 Barbital Sodium 97 185 186 5-( 2-Bromallyl)-5-(l-methyl butyl barbituric acid salt 7 k 152 157 Butabarbital Sodium 100 153..5-151* l 6 k Butallylonal Sodium U5 12? -130 131 -132 Butethal Sodium 35 119 -120 123 -121; Cyclobarbital Calcium 100 l5ii -155 H*9 -150 Dial Sodium 70 163 l6i| -165 Ethyl allyl barbituric acid salt 75 153 -15U 157 Hexethal Sodium 100 m -119 122 -123 Hexobarbital Sodium 100 lUo 139 -HiO Methallatal Sodium 81 155 -156 156 -157 Pentobarbital Sodium 100 120 -121 121 -122 Phenobarbital Sodium 100 165 -169 171 -172 Probarbital Calcium 100 197 -198 198 Prominal Sodium 100 173 -1 7 k 172 Propallylonal Sodium U5 176 -179 178 -179 Rutonal Sodium 73 211; -215 215 Seconal Sodium 93 no crystals 81* -85 Thiopental. Sodium 97 152 -153 153 Vinbarbital Sodium 9 k 152 -153 152 -151*

After determining the effect of $% potassium hydroxide, 20$ acetic

acid and distilled water on barbiturates for 2 k hour periods at room temperature, the same concentrations of acid and alkali and also distilled water were employed to extract tissues known to contain definite quantities of barbiturates* - 1*1- LIVER TISSUE EXPERIMENTS

(a) Digestion of barbiturate-llver mixtures with 5% potassium hydroxide.

Mixtures containing 15 grams of liver and 30 milligrams respectively of each of the barbiturates prepared by using the Waring Blendor were allowed to remain at room temperature for 1* hours to permit absorption of the barbiturates by the tissue. At the end of this period, 30 cc. ali­ quots prepared by adding 5$ potassium hydroxide to the liver specimens containing the various barbiturates were kept at room temperature for 21* hours. The protein material was then precipitated with £$ copper sulfate and filtered. The protein free filtrates, placed in separatory funnels, were acidified with 10$ hydrochloric acid and each solution extracted with chloroform. Four chloroform extracts obtained from each specimen were combined and evaporated on a steam bath. The residues obtained after evaporation of the chloroform extracts, were dried at

1 0 0 ° C for 1 hour, cooled, weighed and the melting points determined.

These results as well as the melting points of the original compounds are shown in Table XVI.

(b) Digestion of barbiturate-liver mixtures with 20$ acetic acid.

Mixtures of liver and barbiturates were prepared the same as in (a) above. At the end of the 1* hour period, 30 cc. aliquots prepared by adding 20$ acetic acid to the liver specimens containing the various barbiturates were kept at room temperature for 21+ hours. The protein material was precipitated with 10$ sodium tungstate. The protein free filtrates, placed in separatory funnels, were acidified with 10% hydro- -1*2-

Table XVI MELTING POIHTS OF BARBITURATES RSCQ7ERED FRQM BARBITPRATE-LIVER MIXTURES AFTER 21* HOUR TREATMENT WITH 5% POTASSIIM HYDROXIDE.

Compound Percent Melting Point Melting Point Mixed with Recovery of Recovered of Original Pure Liver______of Acid Material (°C) Barbiturate (°C)

Allylbarbital 60*3 136 -137 138.>5-139 Allyl-n-butyl barbituric acid 1*1*. 0 no crystals 123. 5-121* Amytal ^0.0 ll*8 *11*9 1U8 -11*8, Aprobarbital 37.3 135 -138 11*2 Barbital 8.0 181 186 5- ( 2-Bromallyl) -5-(1-methyl butyl barbituric acid 102.0 157 157 Butabarbital 57.3 162 -163 161* Butallylonal 60,3 130 -131 131 -132 Butethal 21.0 119 -120 123 -121* Cyclobarbital 30.0 158 -160 11*9 -i5o Dial 52.0 charred 161* -165 Ethyl al3yl barbituric acid 33.6 155. 5-156.5 157 Hexethal 52.3 110 -112 122 -123 Hexobarbital 25.0 charred 139 -11*0 Methallatal 5.0 152 156 -157 Pentobarbital 30.3 120 -121 121 -122 Phenobarbital 15.3 170 -171 171 -172 Probarbital 32.0 192 -19U.5 198 Prcminal 2.2 178 -180 172 Propallylonal 30.3 171 -172 178 -179 Rutonal 5.3 no crystals 215 Seconal 1*0.3 65 - 66 81* - 85 Thiopental 73.0 152 -153.5 153 Vinbarbital 6l.o 152 -153 152 -151*

chloric acid and extracted with chloroform* After combining and evapor­ ating the extracts on a steam bath, the residues were dried at 100°C for

1 hour, cooled and weighed* The melting points of these residues were compared with those of the original compounds and are included in Table XVII. -U3-

Table XVII MELTING POINTS OF BARBITURATES RECOVERED FROM BARBITURATE-LIVER MIXTURES AFTER 2k HOUR TREATMENT -WITH 20# ACETIC ACID*

Compound Percent Melting Point Melting Point Mixed with Recovery of Recovered of Original Pure Liver of Acid Material (°C) Barbiturate (°C)

Allylbarbit al 7U.3 138.5-139 138.5-139 Allyl-n-butyl barbituric acid 55.6 125.5-126 123.5-12U Amytal 88.3 114-7 -1U8 1U8 -1U8.5 Aprobarbital 70.6 1U1.5-1H2 1U2 Barbital 72.3 185 -186 186 #5-( 2-Bromallyl)-5-( 1-methyl butyl barbituric acid 1.0 155 -156 157 BufcEbarbital 72.0 163 -163.5 16U Butallylonal 100.3 128 -129 131 -132 Butethal 75.6 122 -12U.5 123 -12U Cyclobarbital 7U.6 156 -157 1U9 -150 *0131 32.6 163.5 16U -165 Ethyl allyl barbituric acid 36.0 159 157 aHexethal 51.0 123 -1 2 h 122 -123 ■aHexobarbital U7.3 ll;3 -lUU 139 -lUo aMethallatal I4I .0 155 -156 156 -157 Pentobarbital 80.1 121 -122 121 -122 Phenobarbital 91.6 170 -171 171 -172 ■aprobarbital 70.0 198 198 a-Prominal 2.0 170.5-172 172 #Propallylon al 8.0 170 -172 178 -179 Rutonal 90.6 21U -216 215 Seconal 83.0 66 8U - 85 a-Thiopental 12.0 153 -153.5 153 Winbarbital 12.6 151 -152 152 -15U

Crystals of barbiturates were visible in the ground liver after removal of the extract, therefore, this method of extraction can not be used for the quantitative isolation of these compounds*

Since the salts of the barbiturates and in most cases not the free acids are water soluble, we used barbiturate salts in determining the effect of water on these compounds* However, we employed the free acids in our investigations of the effects of acid and base because these are forms in which most of the barbiturates are available to the public* - k k -

In the experiment which follows immediately, the effects of water

on mixtures of tissue and barbiturates were studied* Here the salts 119 were produced by adding sodium hydroxide till a pH of 10 , that of

aqueous solutions of most of the barbiturates, was reached*

(c) The extraction of barbiturate-liver mixtures at pH 10*

Twenty-four mixtures containing 1$ grams of ground liver and 30

milligrams of the respective barbiturates were prepared and allowed to

stand at room temperature for k hours to permit absorption of the barbi­

turates by the tissue* Thirty (30) cc, of distilled water were then added to each mixture and the pH adjusted to 10 with sodium hydroxide*

After standing for 2k hours at room temperature, the mixtures were

filtered and the filtrates treated with $% copper sulfate to remove the protein material which was then filtered. The protein free filtrates

were extracted twice with chloroform to remove some of the impurities,

then acidified with 10% hydrochloric acid and extracted with U success­

ive 1$ cc* portions of chloroform to obtain the barbiturates* Evapor­

ation of the chloroform extracts from each specimen left residues in

each case* Since some of the residues appeared amorphous, all of the residues were dissolved in 10 cc. of 0,1 N NaOH. The resulting solutions

were filtered to remove impurities, acidified with 10% hydrochloric acid,

and re-extracted with U successive V~> cc. portions of chloroform. The purified residues remaining after evaporation of the chloroform were

dried at 100°G for 1 hour, cooled, weighed and the melting points determined* (With the exception of SECONAL, all of the purified barbiturates were crystalline. These values comprise Table XVIII)• -It5- Table XVIII MELTING POINTS OF BARBITURATES RECOVERED FRCM BARBITtlRATE-LIVER MIXTURES AFTER 2 k HOUR TREATMENT AT pH 351 Compound Rercent Melting Point Melting Point Mixed with Recovery of Recovered of Original Pure Liver______of Acid______Acid (°C)_Barbiturate (°C)

Ally lb arbit al £ . 2 138 -139 138.,5-139 Allyl-n-butyl barbituric acid 78.3 122 -123 123.-5-12U Amytal 83.3 1U7 -1U9 1U8 -lit8, Aprobarbital 77.0 lit2 -Ht3 lit 2 Barbital 93.1 185 -186 186 5-(2-Bromallyl)-5-(1-methyl butyl barbituric acid 78.0 152 157 Butabarbital 82.lt 152 -l51t l6it Butallylonal U8.3 128 -129 131 -132 Butethal 39.2 119 -120 123 -1 2 k Cyclobarbital 86.1 153 -155 lit9 -150 Dial 79.9 163 16U -165 Ethyl allyl barbituric acid 77.2 153 -15U.5 157 Hexethal 92.3 118 -120 122 -123 Hexobarbital 93.7 139 -litO 139 -ll+O Methallatal 78.2 155 -156 156 -157 Pentobarbital 91.3 120 -120.5 1ZL -122 Phenobarbital 93.it 166 -169 171 -172 Probarbital 90.6 196 -197 198 Prominal 92.3 171 -173 172 Propallylonal it6.1 176 -179 178 -179 Ru tonal 70.2 213 -211; 215 Seconal 90.1 liquid 8U - 85 Thiopental 93.0 152 -153 153 Viribarbital 90.2 151 -153 152 -15&

It has been noted earlier that color reactions of the barbiturates

are not well defined and that melting points as well should be deter­

mined to establish the identity of the barbiturates# However, an

inspection of Table XIX will show that the melting points of some of the barbiturates are so close together that the identity of the compounds

may be difficult to establish by the melting point alone# For this -US-

Table XIX

MELTING POINTS OF BARBITURIC ACIDS

Melting Point (*G) Name of Barbituric Acid

8H - 85 Seconal 121 -122 Pentobarbital 122 -123 Hexethal 123 -12U Butethal 123.S-12U Allyl-n-butyl barbituric acid 131 -132 Butallylonal 138.5-139 Allylbarbital 139 -lUO Hexobarbital 1U2 Aprobarbital 1U8 -1U8.5 Anytal 1U9 -150 Cyclobarbit al 352 -15U Viribarbital 153 Thiopental 156 -157 Methallatal 157 Ethyl allyl barbituric acid 157 5-(2-Brom allyl)-5-(1-methyl butyl barbituric acid 16U Butabarbital 16U -165 Dial 171 -172 Phenobarbital 172 Praminal 178 -179 Propallylonal 186 Barbital 198 Probarbital 215 Rutonal

reason, it was decided to see if other physical properties of the barbi­ turates could be used to identify them and the refractive indices were next investigated.

Determination'of the refractive indices of the 2 k barbiturates under inve s tigation, ' 1 —

These determinations were made by using a Fisher Refractometer with a melting point heating head, A few crystals of a barbituric acid were placed- on the glass portion of the heating head and covered with a «m»n glass prism* The temperature, controlled by means of a rheostat, was

allowed to rise slowly until the compound had melted and the refractive

index could be measured. It happened that the refractive index could

not be measured at the melting point of the barbiturate, therefore, the

temperature at which a definite reading could be observed on the optical scale of the refractometer was recorded. The refractive indices to­

gether with the temperatures at which the readings were taken are shown

in the following Tables XX (a) through (c)*

Table & (a)

REFRACTIVE INDICES OF BARBITURATES DETERMINED BI THE FISHER REFRACTCMBTBR Refractive Temperature Melting Point Index of at which of the the Melted Refractive Original Pure Barbituric Index was Barbituric Compound Acid taken (°C) Acid (°C)

Allylbarbital 1.1+75 11+3 138.5-139 Allyl-n-butyl barbituric acid 1.1+80 130 123.5-121+ Amytal 1*1+55 168-169 li+8 -21+8 Aprobarbital 1.1+81+ 150 li+2 Barbital 1.5l6 202 186 5-( 2-Bromallyl)-5-(l-methyl butyl barbituric acid 1.1+98 178 157 Butab arbital 1.510 167 161+ Butallylonal 1.500 135 131 -132 Butethal 1*1+70 128-129 123 -121+ Cyclobarbital 1.506 175 11+9 -150 Dial 1.500 176 161+ -165 Ethyl allyl barbituric acid 1.1+81+ 161+ 157 Hexethal 1.516 127 122 -123 Hexob arbital 1. 1+50 11+5 139 -H+o Methallatal 1.531+ 176 156 -157 Pentobarbital 1.1+78 135 121 -122 Phenobarbital 1.530 188 171 -172 Probarbital 1*1+53 213 198 Prominal 1.5o5 193 172 Propallylonal 1.500 191+ 178 -179 Rutonal 1.520 21+5 215 Seconal 1*1+93 95 81+ - 85 Thiopental 1.5o8 171 153 Viribarbital ______^ 1.520 M ...... -i5?_ -iLa, -US- Table XX (a) is arranged alphabetically according to the barbitur­

ates investigated. The same material has been reassembled to produce

Tables XX (b) and XX (c) by arranging respectively according to the

increasing values for refractive indices and for temperatures at which

the refractive indices were determined. These tables may be employed

in the identification of unknown barbiturates when only small quantities

of the compounds are’ available. The melting point can be determined while preparing to measure the refractive index of the same material.

Table XX (b)

REFRACTIVE INDICES OF BARBITURATES DETERMINED BT THE FISHER REFRACTCMETER Refractive Index Temperature at of the Melted which Refractive Barbituric Acid Index was taken(°C) BarbituriclAcid

i.U5o iU5 Hexobarbital ±•163 213 Probarbital 1.U55 168- 169 Jimytal 1.U70 128-129 Butethal 1.U75 1U3 Allylbarbital 1.U78 135 Pentobarbital 1.U80 130 Allyl-n-butyl barbituric acid 1.U8U 150 Aprobarbital 1*U8U 16U Ethyl allyl barbituric acid 1.U93 55 Seconal I.U 98 178 5- (2-Bromallyl) -5- (1-methyl butyl barbituric acid 1.500 135 Butallylonal 1.500 176 Dial 1.500 19U Propallylonal 1.505 193 Prominal 1.506 175 Cyclobarbital 1.508 171 Thiopental 1.510 167 Butabarbital 1.516 127 Hexethal 1.516 202 Barbital 1.520 165 Viribarbital 1.520 216 Rutonal 1.530 188 Phenobarbital 1.53U 176 Methallatal -1+9**

Table XX (c)

TEMISRATtKES AT WHICH REFRACTIVE IM33CES WERE 1 M E M N E D (FISHER REFRACTOMETER)

Temperature at Refractive Index which Refractive of the Melted Index was taken (°C) Barbituric Acid Barbituric Acid

95 1*1+93 Seconal 127 I.$l6 Hexethal 128-129 1.1+70 Butethal

130 1.1+80 Allyl-n-butyl barbituric acid 135 l.i+78 Pentobarbital 3-35 l.$00 Butallylonal

H+3 1.1+75 Allylbarbituric acid U+5 1.1+50 Hexobarbital ISO 1.1+81+ Aprobarbital 161+ 1.1+81+ Ethyl allyl barbituric acid 16$ l.$20 Viribarbital 167 l.$10 Butabarbital 168-169 1 .1+55 Amytal

171 1.508 Thiopental 175 1.506 Qyclobarbital 176 1.500 Dial 176 1.53U Methallatal 178 1.1+98 $- ( 2-Bromallyl)-$- (1-methyl butyl barbituric acid

188 1.530 Phenobarbital

193 i.$o5 Prominal 191+ 1.500 Propallylonal 202 1.516 Barbital 213 1.1+53 Probarbital 21+$ 1.520 Ru tonal

Recently it was found that the presence of sodium cyanide effected a barbiturate determination • In our medicolegal work we have noticed that the presence of cyanide interfered with the identification of some of the barbiturates* Therefore, we decided.to investigate farther.the effect of the presence of cyanide in the detection of barbiturates. -5o-

Tbese experiments were set up to cover a period of three days since

ordinarily at least three days elapse between the time the poison is

ingested by the victim and the analysis is completed by the chemist*

This three day interval allows time for the finding of the deceased by the police or other persons^ the post mortem examination and the actual

chemical analysis*

The estimation of barbiturates in the presence of cyanide*

Twenty-five (25) cc. of solution of each of the 2i+ barbiturates (containing one milligram of a barbiturate and one milligram of sodium

cyanide per cc* of distilled water) were prepared. These solutions were

allowed to stand in the hood for three days at room temperature, and at the end of this time, and still in the hood were acidified with 10#

hydrochloric acid, an excess of 2 cc. of hydrochloric acid being added

in each case. In the case of pure barbiturates, this treatment frees

the barbituric acid, crystals of which are usually preceptible with the naked eye* In six (6) of the barbiturate-cyanide mixtures only, could crystals be seen with the naked eye after the addition of hydrochloric acid. The acidulated solutions were then extracted with U successive

15 cc. portions of chloroform* The residues obtained after the evapora­ tion of the combined chloroform extracts were dried at 100°C for 1 hour, cooled, weighed and the melting points were then determined* These results are given in Table XXI* Table XXI

MELTING POINTS OF MATERIALS RECOVERED FRCM BARBITURATE-CTAMIDE MIXTURES

Precipitate Melting Point Melting Point Visible after of Recovered of Original Pure Barbiturate HC1 Addition Campound(°C) Barbiturate (°C)

Ally lb arbital 128-132 138.5-139 Allyl-n-butyl barbituric - acid . _. 12U-12S 123.5-121* Amytal — ll*8 11*8 -11*8.5 Aprdbarbital — ll*0-ll*l 11*2 Barbital 182 186 5-(2-Bromallyl) -5- (1-methyl butyl barbituric acid + 157 157 Butabarbital — 161* 161* Butallylonal — 132-133 131 -132 Butethal — 121-122 § 123 -HSU 0 Cyclob arbital — 152-153 1 Dial — 165-166 161* -165 Ethyl allyl barbituric acid • m M 155 157 Hexethal + 121-122 122 -123 Hexobarbital — 137-138 139 -11*0 Methallatal + 155-156 156 -157 Pentobarbital + 120-121 121 -122 Phenobarbital — 166-167 171 -172 Probarbital -- 19U 198 Prcminal + 171+ 172 Propallylonal — n h 178 -179 Rutonal •Mm 215 215 Seconal ----- 131-132 81* -85 Thiopental + 155 153 Vinbarbital 157 152 -151*

Our final investigations were made to determine the stability of the solid barbiturates to air and atmospheric moisture in the presence of light and heat, since there appears to be some uncertainty on this point • -52-

Determination of the stability of solid barbiturates*

The barbituric acids were exposed to the atmosphere at room temper­

ature for 60 days* At the end of this time* the melting points of these barbituric acids were compared with those of the original pure barbituric

acids* These results are given in Table XXII*

Table XXII

MELTING POINTS OF BARBITURIC ACIDS PRIOR AND AFTER EXPOSURE TO AMCSPHERIC CONDITIONS AT RQCM TEMPERATURE

Melting Point (°C) of Melting Point (°C) Barbituric Acid after of Original Pure Barbituric Acid______60 Day Exposure______Barbituric Acid

Allylbarbital 138,.5-139 138,.5-139 Allyl-n-butyl barbituric acid 119 123..5-12U Amytal lii-7 —124.8 1U8 - m Aprob arbital 139 -1U0 1U2 Barbital 186 -187 186 5-(2-Bromallyl)-5-(1-methyl butyl barbituric acid -156 157 Butabarbital 16 3 I6I4. Butallylonal 129 -130 131 -132 Butethal 122 -123 123 -1 2 k Cyclobarbital 151 -i£2 Hi9 -150 Dial 16k -166 1 6k -165 Ethyl allyl barbituric acid 160..5 157 Hexethal 120 -121 122 -123 Hexobarbital liiO —li|2 139 -litO Methallatal 155 -156 156 -157 Pentobarbital 113 -119 121 -122 Iftenobarbital 171 -173 171 -172 Probarbital 199 -200 198 Prominal 17U -175 172 Propallylonal 178 -179 178 -179 Rutonal 215 -216 215 Seconal 83 - 8U 8U - 85 Thiopental 152 9 CO 153 Vinbarbital 157 ..... & -151* Several salts of the barbituric acids left exposed for the same period of time were examined visually and all appeared unchanged with the exception of HEXOBAHBITAL SODIUM. -53- DISCUSSION

The percent recovexy of barbiturates subjected to treatment with

potassium hydroxide and recovered by extraction of the acidified salts

with chloroform ranged from lu8 to I0lu5 as shown in Table XIII.: BAB**

BITAL And RUTONAL, two of the long acting barbiturates, were among the

compounds which apparently decomposed during the alkali extraction pro­ cess* The percent recovery of BARBITAL was 8*b while that of RUTONAL

was 8*6* t w o other compounds which seemed to have been effected by the action of the alkali were METHALLATAL with 6*8 percent recovery and FROM INAL with a U*8 percent recovery* Three of these four compounds,

BARBITAL, FROM INAL and METHALLATAL have ethyl groups as one of the side chains* There is no other similarity in the Structure of these compounds,

therefore, it seems unlikely that the results are due to a particular

structure* Seventy-five percent of the acids recovered after the alkali treat­ ment had melting points different from the original barbituric acids* With the exception of CYCLOBAR BITAL, DIAL, HEXOBARBITAL and PROW INAL, the melting points of the acids in this seventy-five percent were the same or lower than those of the corresponding original compounds* The melting points of six of the barbiturates (AMYTAL, 5-(2-BRCMALLSL)-5-(1-

METHYLBUTYL) BARBITURIC-ACID, BUTALLYLONAL, PENTOBARBITAL, PROBARBITAL and THIOPENTAL) were unchanged by treatment with alkali* There is no correlation between the structure of the compounds the melting points of which were unchanged and the percent recovexy of these six compounds*' Two compounds, ALLIL-n-BUTXL BARBITURIC ACID AND RUTONAL, after treatment with 5 % potassium hydroxide and recovexy of the material from an acidified solution, yielded products which had no crystalline struc­ tures.'1 Here again, there seemed to be no correlation between the structure of the compound, the percent recovery of these two acids or -A- thelr melting points*

The 10U.5 percent recovexy of 5>- (2-BRCMAlLXL)-£- (1-METHHBDTIL)

BARBITURIC ACID may have been due to partial oxidationy the behavior did not correspond to that of the other bromallyl derivatives, BUTALLIL-

CNAL and FRGPALLILONAL, when treated in ths same fashion*

It has now been established that all of the 2k barbiturates studied may be recovered in varying quantities after treatment with potassium hydroxide* Occasionally the recovered barbituric acid may not be in crystalline form*

The recovexy of barbituric acids after treatment with 20% acetic acid was better than after treatment with alkali* Table XIV shows that the percent recovexy ranged from 38*0 to 10U*l* Compounds marked (*) in

Table XIV were visibly insoluble or only partially soluble in 2056 acetic acid*1 The recovery of such compounds ranged from 71*256 for METHALLATAL to 102.356 for VINBARBITAL*

It would seem that even if a barbiturate were insoluble in 2056 acetic acid, a chlorofonn extraction

5-(2-BR(MALLTL)-5»(l«MEmrLBUTrL) BARBITURIC ACID and VINBARBHAL were completely recovered*" On the other hand, BUTABARBITAL, which was completely soluble in 2056 acetic acid, was among the compounds which yielded a high percent recovery (10U56). In this case, the k% increase in weight was probably due to some degree of oxidation**

It appears, therefore, that no correlation can be made between the solubility of the barbituric acids in 20$ acetic acid and the extent to

which they are recovered ty extraction with chloroform*!

All of the materials recovered after the 2h hour treatment of the

barbiturates with 20% acetic acid had melting points which were somewhere

within the range of those of the original barbituric acid, with three

exceptions, CYCLOBARBITA1, FROPALLYLONAL and SECONAL, CXCLOBARBITAL had

a melting point higher than the original acid while FROPALLYLONAL and

SECONAL, had a melting points lower than the original barbituric acids*

It has now been established that all of the 2b barbituric acids

studied can be recovered to some extent following 2h hour treatment with

20% acetic acid*

The behavior of the various barbituric acid salts allowed to stand

for 2h hours in distilled water is shown in Table XV* At the end of the

2k hour period, 100$ of nine of the barbituric acid salts studied was re*

covered*' The recovery of other barbiturates ranged from 35$ for BUTETHAL

SQDUM to 97$ for BARBITAL SODIUM • There appeared to be no correlation between the percent recovery of the barbituric acid and its structure*1

In no case was the percent recovexy over 100$* SECONAL was the only barbituric acid which could not be recovered in crystalline form after

treatment with distilled water for 2k hours at room temperature*'

These results indicate that salts of barbiturates are stable in water for at least 2b hours moreover, when thB acids are freed from the salts and extracted with chlorofoim, crystalline residues may be obtained after evaporation of the chloroform except in the case of SECONAL*)

SECONAL, CXCLOBARBITAL and BUTABARBITAL were three compounds which possessed melting points which did not compare favorably with the original compounds (Table XV)* SECONAL crystals could not be obtained;

BUTABARBITURIC ACID had a lew melting point while the melting point of

CYCLOBARBITORIC ACID was too high*

It may then be concluded th^t aqueous solutions of the salts of

barbituric acids are more stable over a 2k hour period than barbituric

acids in $% potassium hydroxide or 20% acetic acid* The quantity of barbiturates recovered from the tissue by a 2k hour

extraction with $% potassium hydroxide ranged from 2,2% far FRCMINA1 to 1G2*0/6 far 5-(2-BR

Da one or two cases, the results were almost similar to those obtained when the pure compounds were treated with 5>j6 potassium hydroxide, but in the majority of cases the results were slightly less*

The quantity of barbiturates recovered from the tissue by a 2k hour

extraction with 2056 acetic acid ranged from 1*056 for 5-(2*»BRCMALLYL)-5^1

-METHYLBUTTL) BARBITURIC ACID to 100.3/6 for BUTALLYLONAL (Table XVII). Barbiturates extracted from tissues by this method had a wider melting

point range in some cases than the barbiturates treated in a similar manner in the absence of tissue (Table XIV)* With the exception of

ETHIL ALLYL BARBITURIC ACID, the barbiturates which were not recovered

in appreciable quantities were the compounds which were not completely

soluble or were insoluble in 2056 acetic acid. Da some instances the

extracted tissues still, contained visible crystalline material. Da all probability, the presence of these crystals in the extracted liver after

extraction would account for the apparent loss of the barbiturate.'

Since the pH of aqueous solutions of salts of the barbiturates is XX9 believed to be between 9 and 10 , we carried out the extraction of the barbiturates from tissues at this pH* The recovexy under these conditions (39*2-93.7/6) compared favorably with that obtained when pure salts were allowed to stand in water, acidified and extracted with chloroform

(35-lOOjC). The poorest recovexy (39.2J6) was greater than either with the acid or the alkali treatment*1 There was no apparent correlation between the percent recovery and the structure of the compound*1 With the exception of BUTABAEBITAL and SECONAL, the melting points of the compounds recovered after this treatment compared favorably with that of the pure barbituric acids* SECONAL* as usual, was a viscous liquid*1

The melting point of BUTAEARBITAL was about 10 degrees too low*1

Table XIX shows that PENTOBARBITAL (nwp* 121-122*C); HEXETHAL (m.p.

122-123°C)j BUTETHAL (nup. 123-12i*°C) and ALUL-n-BUTIL BARBITURIC ACID

(m.p. 123.5-12R°C) melt within a very narrow range, however, it may be seen in Table XX (a) that the refractive indices of these four compounds are far enough apart so that these four barbiturates may easily be identified; PENTOBARBITAL m.p. 121 -122*C R*I* UHftxsB HEXETHAL m.p. 122 -123°C R.I. l.£l6X87 BUTETHAL uup* 123 •12U°C R.I. l*H70xae-129 ALLTL-n-EUTTL BARBITURIC ACID m.p. 123.5-12R“C RalV l.U80X3O

The following compounds may be identified in a similar manner, using melting points in conjunction with the refractive indices*

ALLYL BARBITAL m.p*1 138;£-139°C R.I* l.U?SX43 HEXOBARBITAL HUp*1 139 *Hl0*C R.I. 1*160X46 APROBARBITAL m.p. ll*2°C R.I. 1.1*81^,50

BUTABARBITAL m.p. 16U*C R*I* l.£l0167 DIAL m.p. 161; —165>*C R.I. l.J?00X7g

PHEN0BARB1TAL m.p. 171 •172* C R.I. l.£30X88 PR®INAL nup. 172 R.I. l.£0£x98

There are also a number of barbiturates (7) which have melting points between 11*8 and 15>7*C* These compounds likewise may be differen­ tiated by means of tha refractive indices. They are* -58-

AMYTAL m.p. lU8-ll*8.5°C R.I. l.U5516Q l69 CYCLOBARBIT AL m.p. lltf-l^CTC R.I. 1.5o617s V INBARBITAL m.p. l52-l5U°C R.I. 1,520^5 THIOPENTAL m.p.X53°C R.I. 1.5o8i7i METHALLATAL m,p. 156-157 °C R.I. 1.53i|i7Q ETHYL ALLYL BARBITAL m.p. l57°C R.I. l.U8i|l84 5~( 2-BRCM ALLYL) -5~(l’*IETHYL BUTYL BARBITURIC ACID m.p. l57°C R.I. l.U98lva

Although ETHYL ALLYL BARBITURIC ACID and 5-(2-BRCMALLYL)-5-(l- METHYLBUTYL) BARBITURIC ACID have the same melting point, the temper­

atures at which the refractive indices are observed are different as

indicated by the subscripts. ETHYL ALLYL BARBITURIC ACID (m.p.

R.I. l.i4.8Ul84)j 5-(2-BRCMALLYL)-5-(1-METHILBUTYL) BARBITURIC ACID (m.p. 157 C$ R.I. I*l4-98^7g).

The remaining six compounds investigated had melting points which were far enough apart for their identification, nevertheless, the refractive indices of these barbiturates were determined and recorded in Tables XX (a) through (c). Investigations of the effect of the presence of cyanide on the determination of barbiturates suggest the formation of some type of barbiturate-cyanide complex. Filter paper strips moistened with 0.2$ alcoholic guiaic solution and 0.1$ aqueous copper sulfate did not give any definite indication of the evolution of hydrocyanic acid when held over the neck of the flasks after the barbiturate-eyanide mixture was acidified. A positive reaction would have been indicated if the moistened filter paper strip had turned blue. After the barbiturate- eyanide solutions were acidified and an excess of 2 cc. of 10$ hydro­ chloric acid added to each, only six (6) of the mixtures yielded cloudy solutions as do pure barbiturate solutions. Pure barbiturate solutions -59- yield cloudy* solutions upon acidification if their salts are present in a concentration of 1 mg* per cc* of water* Even though the free barbituric acid was not visible as a precipitate after the addition of 1 0 % hydrochloric acid to the barbiturate-eyanide solutions, crystalline substances remained in every case after evaporation of the chloroform extract* Melting points run on these residues ranged from 10° below to 1|S*C above those of the original barbiturates

(Table XXI)*

With the exception of THIOPENTAL, all of the compounds which ex­ hibited an increase in melting point were unsaturated compounds* These barbiturates were: ALLIL-n-BDTXL BARBITURIC ACID} BUTALLYLONAL;

CYCLOBARBITAL} HEXETHAL; FRCMINAL; SECONAL and VINBARBITAL* There were, however, some unsaturated barbituric acids which yielded compounds with lower melting points than the original respective barbiturate and still others in which the melting points did not change at all* SECONAL, which is usually viscous upon recovexy from chloroform, was crystalline in this instance* CYCLOBARBITAL; SECONAL and V INBAR BITAL were three compounds with increases of 3°C or more in the melting points* Among the recovered barbiturates with melting points little altered were:

ALLYL-n-BUTIL BARBITURIC ACID; JMYTAL; APROBARBITAL; 5-(2-HRCMALLYL)-5-

(1-METHYLBUTXL) BARBITURIC ACID; BUTALLYLONAL; BUTETHAL} DIAL; HEXO­

BARBITAL; PENTOBARBITAL; PROS!INAL and RUTONAL*'

Da the barbiturate procedure when the barbiturate-eyanide mixtures were acidified with lo£ hydrochloric acid to pemit the extraction by chloroform, all of the eleven (1 1 ) compounds just mentioned appeared to -60-

be physic ally changed since they did not form cloudy solutions as is

usually the case when hydrochloric acid is added* If our theory

that these barbiturates formed complexes with the cyanide in the eleven

cases is correct, then we may possibly explain the fact that the melt*

ing points remained unchanged by one or the other of the following

assumptions, the first one of which seems to be more likelyt

1* The barbiturate-eyanide complex was destroyed during removal

of the chloroform or the subsequent 1 hour drying at 1 0 0 °C, or

2* The barbiturate-eyanide complex has the same melting point as

the respective barbiturate*

None of the barbiturates used in the barbiturate-eyanide experiment which yielded a cloudy solution upon acidification with hydrochloric acid showed an appreciable increase in melting point* These six (6 ) barbiturates which yielded cloudy solutions when acidified were: 5 *

(2-BRCM ALLYL)-5-(l-KE'fflYL'BUiYL) BARBITURIC ACID} HEXETHAL} METHALLA-

TALj PENTOBARBITAL; FRCMINAL and THIOPENTAL* The melting points of the six recorded barbiturates did not change appreciably over those of the respective pure compounds*

Since the guiaic*copper sulfate-filter paper test did not indicate the presence of free hydrocyanic acid in this instance, after acidifi­ cation of the barbiturate-eyanide reaction mixtures, it can be said that six (6 ) of the barbiturate-eyanide reaction mixtures behaved as barbiturates under similar conditions* Li this group of six barbi­ turates, FRCMINAL and THIOPENTAL were the only two which yielded compounds with a melting point 2° or more above that of the original compound* -&U

A determination of the carbon content of these extracted compounds

might indicate whether or not the cyanide had remained in combination

with the barbituric acid*

If the barbiturates deteriorate by long exposure to the atmos­

phere, this fact was not apparent by the melting point, with the

possible exception of V IN BARBITAL (Table XXI}* It would seem that a

reaction at the double bond in the unsaturated barbiturates or oxida­

tion of the side c ha ink) at one of the terminal carbon atoms would

be indicated by a noticeable change in the melting point. At the end

of 60 days HEXOBARBITAL SODUM was the only barbituric acid salt, which upon a visual observation, appeared to have picked up moisture*

In order to cover the field of Forensic Chemistry 3 s applied to barbiturates, representative products as they appear on the open market were used in these investigations*

We gratefully acknowledge gifts of thB following barbiturates which were used in this investigation:

ALLYL-n-BUTYL BARBITURIC ACID; BUTETHAL? ETHYL ALLYL BARBITURIC

ACID; MOSIBAL* PENTOBARBITAL; PHENOBARBITAL and PENTOTRAL from Abbott

Laboratories^

NOSTAL, PERNOSTON and S2EM0BAL from Ames Company, Inc*

DIAL from Giba Pharmaceuticals, Inc*

ALTJRATE from Hoffraan-La Roche, Inc*

AMOBARBITAL and SECOBARBITAL from Eli Lilly and Company*

RUTONAL from Hay and Baker Ltd, Dagenham, England

BUTISOL from McNeil Laboratories, Ihc*

ORTAL from Parka, Davis and Company • 6 2 -

SANLOPTAL from Sandoz Pharmaceuticals DELVINAL from Sharp and Dohme, Inc*

IPRAL from E. R* Squibb and Sons, and

CYCLOBARBITAL5 EVTPAL and MEPH OB ARB IT AL from Winthrop-Stearnsi* Inc* -63- SUMMABJ

Twenty-four barbiturates were tested for their stability in %% potassium hydroxide (Table XIII) and in 20% acetic acid (Table XIV) for

2k hours at room temperature. The stability of the salts of this sane series of barbiturates in distilled water for 2h hours at room temper­ ature was also investigated (Table XV). Tissues containing known quantities of the various barbiturates were extracted by three methods:

1. With potassium hydroxide for 2b hours at room temperature (Table XVI).

2. With 20% acetic acid for 2k hours at room temperature (Table XVII)

3. At pH 10 (corresponding to that of salts of barbituric acids in

distilled water) for 2h hours at room temperature (Table XVIII).

Refractive indices Tables XX(a) through (c) of the series of barbiturates were determined by use of a Fisher Refractometer with a melting point heating head. The readings were taken at temperatures at which the refractive indices could best be observed and not at the transition points. The temperatures at which the readings were taken are recorded in Table XX(c).

A study of barbiturates in the presence of sodium cyanide was made.

There was no apparent correlation between the type of reaction and the structure of the compound. The fact that none of the barbiturate- eyanide mixtures yielded hydrocyanic acid when acidified with hydro­ chloric acid (as indicated by the negative guiaic-copper sulfate-filter paper test) would seem to suggest that all of the barbiturates had reacted with sodium cyanide; however, the melting point of the reaction product was not enough evidence that the product was a barbiturate- eyanide addition complex* In 75$ of the barbiturates investigated, physical evidence was indicated by the following observations: (1 ) the addition of hydrochloric acid to the barbiturate-eyanide mixtures did not produce cloudy solutions and (2) the negative guiaic-copper sulfate filter paper test. Barbituric acids and their respective salts were exposed to the atmosphere for 60 days at room temperature* The melting point of

VINBARBITAL increased. Melting points of the other barbituric acids remained the sane* that is, within the same range* HEXOBARBITAL SODIIM was the only salt which, by visual observation, picked up moisture to any degree in 60 days*

CONCLUSIONS

The results of this investigation seem to justify the conclusion that the extraction of barbiturates' from tissues with aqueous solutions at pH 10 is the method of choice for Forensic Chemistry*

Comparison of the results obtained by extraction of barbiturates from tissues treated with 5$ potassium hydroxide or 20$ acetic acid for

2k hours with the results obtained by extraction of barbiturates from solutions of 5$ potassium hydroxide or 20$ acetic acid after standing

2h hours showed little differences*

Refractive indices of melted barbiturates may be used as an aid in the identification of the compounds*

Aqueous solutions of salts of barbituric acids do not deteriorate -65- appreciably during a 2 k hour period*

Sodium cyanide interferes with the identification of barbiturates to some extent*

With the exception of VINB ARBITAL and HEXOBARBITAL SODIUM, exposure of barbiturates and their respective salts to atmospheric conditions apparently does not cause deterioration of the compounds* -66-

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autobiography

I, Gwendolyn Bertha Carson, was b o m in Berkeley, California,

October 25, 1918* I received my secondary school education at South

High School in Columbus, Ohio* Both my undergraduate and graduate training to date have been received at The Ohio State University* The Bachelor of Science degree in Bacteriology was conferred in 19U3 and the Master of Arts degree with Physiological Chemistry as my field of specialization in 1?U5« While in residence, I acted in the capacity of part-time Technical Assistant in the Department of

Physiological Chemistry* From 19ll5-1950, I held a full time appoint­ ment as Technical Assistant specializing in Toxicology under the supervision of Dr. Clayton S* Smith* In this capacity, I analyzed foods, medicaments and various biological specimens for doctors, hospitals, coroners and law enforcement officers throughout the State of Ohio* In 1?50, I received an appointment as Instructor at The

Ohio State University, where I have continued my previous services in addition to teaching laboratory courses in Toxicology, Food Analysis and Blood Analysis* Since the language requirements and most of the course work had been completed before I became an Instructor, I was permitted to carry on my work on the dissertation while .holding this appointment*