Arch. Toxicol. 34, 77---80 (1975) 9 by Springer-Verlag 1975

Two Unusual Barbiturate Deaths A l p h o n s e P o k l i s a n d Ali Z. H a m e l i State Toxicology Laboratory, State University Station, Fargo, l~orth Dakota and Forensic Sciences Laboratery, Wilmington, Delaware, USA Received April 8, 1975

Abstract. The case histories and toxicological examination of two unusual fatal barbiturate poisonings are presented. One case involved the suicidal ingestion of a veterinarian euthanasia preparation containing pentobarbital which resulted in extremely high concentrations of the drug in the blood and liver. The second case involved the suicidal ingestion of vinbarbital, a drug no longer available on the american pharmaceutical market. The case exemplifies the necessity for definitive and analytical procedures for the identification of drugs in biological samples. Identification of pentobarbital and vinbarbital was by infrared spectrophotometry and thin layer chromatography. Quantitation of these drugs was by ultraviolet spectrophotometry. Key words: Pentobarbital - - Vinbarbital - - Fatal Poisoning - - Infrared Spectrophotometry - - Distribution. Zusaramen/assung. Zwei auBergew6hnliche Todesf~lle dureh Barbiturs~urepr~parate. Die Krankengeschichten und die toxikologischen Untersuehungen yon zwei auBergew6hnlichen tSdlichen Barbiturs~urepr~paratvergiftungen werden in dieser Arbeit vorgelegt. Ein Fall betrifft die selbstmSrderisehe Einnahme eines veterin~ren EuthanasieprKparats, das Pentobarbital enth~lt. Dies verursachte sehr hohe Konzentrationen im Blur und in der Leber. ])or zweite Fall betrifft die selbstmSrderische Einnahme yon Vinbarbital, das am amerikanischen pharmazeutisehen Markt nicht mehr erhKltlich ist. Dieser Fall dient als Beispiel fiir die Notwcndigkeit yon definitiven, analytischen Verfahren fiir die Idcntifizierung yon Fremdstoffen in biologischen Proben. Die Identifizierung yon Pentobarbital und Vinbarbital wurdc (lurch Xnfrarot-Spektrophotometric und Dilnnachieht-Chromatographie erzielt. Zur quantitativen Analyse wurde die UItraviolett-Spektrophotemetrie eingesetzt. ~qchli/tsselw6rter: Pentobarbital - - Vinbarbital - - Vergiftung - - Inffarot-Spektrophotemetric - - Verteilung.

Case Histories Case 1. In December 1973, the body of a 53 year old, white male was found in a motel. The deceased, a veterinarian on his way to a professional convention, had registered at the motel on a Friday evening. None of the motel's employees observed the deceased over the weekend and as a result on Monday morning the management sent a cleaning lady to check on the deeeased's well being. She discovered the door to the deeeased's room was locked and bolted and a foul odor was eminating from the room. A maintenance man was called and after a forced entrance he found the deceased lying naked on the bed. A bottle of bourbon whisky was on the night stand next to the bed. A glass with a residue in the bottom and an uncapped empty 250 milliliter bottle of "Toxital" was discovered in the trash basket. "Toxital" is a veterinarian euthanasia preparation made by Jensal Laboratories used to painlessly destroy sick or injured animals. I t contains 178 milligrams of sodium pentobarbital per milliliter in

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Alphonse Poklis and Ali Z. Hameli

50 % propylene glycol base. The body was removed to the Office of the Chief Medical E x a m i n e r a n d a n autopsy was preformed. The pathological findings were unremarkable. Suicidal ingestion of "Toxical" was suspected as the cause of death; therefore tissues and fluids were sent to toxicology laboratory for analysis. Case 2. I n October of t973, a 78 year old, white female was found dead in her bed at home. A local telephone service which daily called elderly persons to inquire as to their health or needs, h a d received no answer to a call to the deceased's residence. The agency then notified the state police who subsequently discovered the deceased clad in a slip and sweater lying in bed a n d covered to the waist b y two blankets. A suicide note and a will were found near the body. The will specified particular items of clothing in which the deceased wished to be buried. All these clothes were on a hanger outside one of the closets near the bed. A cleaning lady stated the deceased was particularly depressed each year a t this time as it was the anniversary of her h u s b a n d ' s death. A family doctor states t h a t several years prior the deceased h a d a t t e m p t e d suicide with barbiturates a n d also t h a t she supported a local euthanasia society. Containers for eighteen different prescription drugs were found in the home. These drugs included chloral hydrate, methylprylon, mcprobamate, propoxyphene a n d imipramine. Also uncovered was a 19 year old prescription dated J u l y 1, 54 and labeled Delvinal| 0.1 (vinbarbital). This drug has not been available on the american pharmaceutical m a r k e t for over 12 years. The body was removed to the Office of the Chief Medical E x a m i n e r and a n autopsy was performed. The pathological findings were unremarkable and tissues a n d fluids were sent to toxicology laboratory for analysis.

Toxicological Examination Sto'mach Contents. 5 g of stomach contents were mixed with 5 ml of dilute HCI and extracted with 3 50 ml portions of diethyl ether. The ether fractions were combined, then washed with l 0 ml of distilled water. The ether was filtered through anhydrous Na2SO 4 and evaporated to dryness on a steam bath. A portion of the white residue was incorporated into a K B r disc and the infrared spectrum was recorded against a n air reference. The infrared spectrophotometric determinations were performed on a Pcrkin Elmer Model 457A. Extraction Procedure. 100 g of liver and 50 g of kidney were screened for drugs and poisons b y steam distillation followed b y a continuous extraction procedure (Freimuth, 1960). Strong acid, weak acid, neutral, basic a n d amphotcric (saturated bicarbonate) extract fractions were collected and subjected to t h i n layer chromatography. Thi.~ Layer Chromatography. Determinations were made using 0.25 m m thick silicagcl GF plates. The acid and neutral fractions were developed in two systems; chloroform: acetone (9 : 1) a n d chloroform: butanol : a m m o n i u m hydroxide (2t0: t20:15). The drugs were localed by two spray systems; exposing the plate to chlorine gas followed b y O-toluidine spray, and spraying with 0.01% diphenylcarbazone followed by 0.25 % mercuric sulfate. The basic and amphoterie fractions were developed in a system of b e n z e n e : d i o x a n e : e t h a n o h a m m o n i u m hydroxide (150:i20:15:15). Spots were visualized b y spraying with 0.01% fluoreseamine in acetone, followed by ethanolic sulfuric acid, potassium iodoplatinate solution and Dragendorfs solution. Gas Chromatography. In both cases, ethanol a n d also in case 1, propylcne glycol, were determined in blood b y gas chromatography (Gupta et al., 1972). The analyses were preformed on a 50 cm • 0.6 cm u-shaped glass tube packed with Chromosorb 102, 60/80 mesh. The oven t e m p e r a t u r e was t 5 5 ~ C a n d the flame ionization detector temperature was 250 ~ C. The gas flow rates were; helium carrier gas 30 ml/min, hydrogen 30 ml/min, and air 240 ml/min. The resulting retention times were: ethanol 60 sec, propylene glycol 175 sec and isopropanol (internal standard) 84 sec. Barbiturate Quantitation. In each case, once the b a r b i t u r a t e was identified, quantitation of the drug in blood, urine, a n d aliquots of the tissue extracts was done b y differential ultraviolet spectrophotometry (Broughton, 1956). I n all determinations the maximums, minimums, a n d isobestic points were in agreement with those of s t a n d a r d pentobarbital and vinbarbital solutions. Tissue extracts were clean enough to permit direct u.v. determinations, chromatographic purification being unnecessary.

Two Unusual Barbiturate Deaths

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Table I. Tissue Concentrations of Barbiturates in mg %

Tissue Stomach Contentsa Blood Liver Kidney Urine

Case I

Case 2

( P entobarbital) 2.t 15 32 6.3 --

( V inbarbital ) 0.38 5.3 16.4 8.5 8.0

Expressed as total barbiturate present, in grams.

Results and Discussion Case 1. Pentobarbital, propylene glycol and ethanol were detected in the tissues and fluids of the deceased. The drinking glass recovered from the bedside trash can and the stomach contents contained pentobarbital and propylene glycol, indicating the deceased had drunk "Toxital". The blood contained 0.06% (w/v) ethanol, 20 rag% (w/v) propylene glycol and i5 rag% pentobarbital. The tissue concentrations of pentobarbital are presented in Table I. A fatal blood level of pentobarbital is generally accepted as above 2 rag%. The highest pentobarbital blood level reported is i i . 2 rag% (Rehling, 1967). To check the results of the present case, duplicate analyses were preformed on 5 ml and t ml of blood. The analysis gave a blood level of 15.2 m g % +_ 0.3 rag%. The absorbed dose, based solely on the blood volume, and liver and kidney weights, is approximately 1.5 g of pentobarbital. This, combined with the amount left in the stomach, accounts for 21 ml of the " T o x i t a l " preparation. The high pentobarbital blood level in this case is the result of the massive quantity of the drug which was easily ingested in a vehicle conducive to rapid absorption. One hundred milliliters of "Toxital" contains 17.8 g of sodium pentobarbital. Case 2. Vinbarbital and ethanol were the only drugs detected in the fluids and tissues of the deceased. The blood ethanol level was 0.04% (w/v) and the barbiturate concentrations are presented in Table I. The unusual feature of this case is the use of a 19 year old prescription drug as the suicidal agent. Because vinbarbital has not been used in america for over a decade, we had no reference data of our own on this drug. Fortunately a few capsules of Delvinal| were recovered at the scene. An infrared spectrum of the residue from the stomach contents was identical to the spectrum of the extracted capsule material and consistent with data in a standard reference (Clarke, t969). The bands of greatest intensity in the infrared were at wave numbers 1370, 1675 and i760. The melting point of the capsule extracted vinbarbital was 160 to t63 ~ C and the melting point of the stomach residue was 159 to 162 ~ C. The R f ' s of the phenobarbital and vinbarbital are not significantly different in our two acid TLC systems, but we distinguish vinbarbital b y spraying with 0.i % potassium permanganate solution. Vinbarbital gave a yellow color while phenobarbital was uureactive. This ease points out the necessity for toxicology laboratories to have a wide range of on-hand reference data for m a n y compounds. I t also exemplifies the need for sound eonformational procedures. H a d only thin layer chromatography and

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u l t r a v i o l e t s p e c t r o p h o t o m e t r y been preformed i n this ease, p h e n o b a r b i t a l m a y well h a v e b e e n identified as the suicidal agent. The m e l t i n g p o i n t a n d a n f r a r e d s p e c t r u m p r o v i d e d t h e correct identification.

References Broughton, P. M. G.: A rapid ultraviolet speetrophotometrie method for the detection, estimation and identification of barbiturates in biological material. Biochem. g. 63, 207 to 213 (1956) Clarke, E. G. C. : Isolation and identification of drugs, pp. 569 and 594. London: The Pharmaceutical Press 1969 Freimuth, It. C. : Isolation and separation of poisons from biological material. In: Toxicology mechanisms and analytical methods, vol. I (ed. C. P. Stewart, A. Stolman), pp. 285--302. New York: Academic Press 1960 Gupta, R. N., Galdenzi, S., Keane, P. M. : The determination of plasma ethanol by gas liquid chromatography using trichloracetic acid as the protein precipitant. J. forens. Sci. 17, 453--455 (1972) Rehling, C. J. : Poison residues in human tissues. In: Progress in chemical toxicology, Vol. 3 (ed. A. Stolman), pp. 363--386. New York: Academic Press Alphonse Poklis State Toxicology Laboratory State University Station Fargo, North Dakota 58102 USA

Ali Z. Hameli Chief Medical Examiner and Director of Forensic Sciences Laboratory 200 South Adams Street Wilmington, Delaware 19801 USA