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Suicide by injection of a veterinarian barbiturate euthanasia agent: report of a case and toxicological analysis
Forensic Science International 131 (2003) 103±107
Suicide by injection of a veterinarian barbiturate euthanasia agent: report of a case and toxicological analysis Nathalie Romain*, Christian Giroud, Katarzyna Michaud, Patrice Mangin Institut Universitaire de MeÂdecine LeÂgale de Lausanne, rue du Bugnon 21, 1005 Lausanne, Switzerland Received 10 July 2002; received in revised form 20 October 2002; accepted 6 November 2002
Abstract The history and toxicological ®ndings of a suicidal case involving injection of a veterinarian barbiturate euthanasia agent (Vetanarcol1) containing pentobarbital are presented. Blood pentobarbital concentrations compatible with drug overdose were determined. Almost identical levels were found in blood, cerebrospinal ¯uid (CSF) and vitreous humour (VH). The highest concentration was measured in the bile. The present case is compared with similar rare cases in the literature. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Suicide; Barbiturates; Pentobarbital; Euthanasia; Veterinary
1. Introduction Vetanarcol1 and T-611 are two pharmaceutical preparations very commonly used for the euthanasia of animals. Vetanarcol1 has been marketed in Switzerland since 1957 and is available in 100 ml bottle with each millilitre containing 162 mg of pentobarbital sodium-salt, 20 mg of benzyl alcohol as preservative and 500 mg of propylene glycol as vehicle. Depending on the dose, it is used by intravenous or intra-peritoneal administration for the animals narcosis or euthanasia. In the last case, it produces rapid central nervous system depression and cardiovascular collapse [1]. For instance, a dog is rapidly killed by injection of 0.5±1.0 ml/kg of Vetanarcol1. Another common procedure is to administrate Vetanarcol1 as a premedication before a lethal dose of T-611. T-611 is a mixture of three compounds containing embutramide, a general anaesthetic, mebenzonium, a muscle relaxant, and tetracaine, a local anaesthetic [2]. According to the Swiss law, pentobarbital is a controlled substance. Vetanarcol1 can be only bought by veterinary surgeons with a state licence for narcotics.
* Corresponding author. Tel.: 41-21-3147070; fax: 41-21-3147090. E-mail address: [email protected] (N. Romain).
On the one hand, accidental and volunteer overdoses caused by short-acting barbiturates in humans have become rare since these molecules became commercially unavailable as sleeping drugs/hypnotics [3]. On the other hand, suicidal ingestion of a lethal dose of pentobarbital has increased because its use is advocated by the ``Exit'' association. This association provides assistance to individuals wishing to commit suicide because of lasting painful disease or severe degradation of their physical condition. Infrequent suicides with pentobarbital in medical and paramedical profession, have also been reported. We report a fatal deliberate overdose attributed to Vetanarcol1. The toxicological ®ndings are presented and compared to those published in the literature. 2. Case history A 51-year-old man, administrator of the county society for the prevention of cruelty to animals was found dead in his apartment next to the refuge. At the scene of death, a 100 ml bottle of Vetanarcol1 containing about 2 ml of liquid, with an empty 10 ml syringe ®xed in its cap was found close to the body. Several farewell letters were discovered on the harddisk of his computer. The victim showed an abdominal ecchymosis with two punctured wounds. The autopsy was carried out 24 h after the discovery of the corpse and there was no feature of decomposition. The
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N. Romain et al. / Forensic Science International 131 (2003) 103±107
body weighed 69 kg. The right side of the abdomen showed a large (18 cm 10 cm) purple ecchymosis with two punctured wounds which could be considered as injection marks. No other traumatic lesion was noticed. Except a diffuse visceral congestion, autopsy ®ndings were unremarkable. Microscopic examination of organs showed no signi®cant pathologic changes. Suicidal administration of Vetanarcol1 was suspected as the cause of death. For toxicological investigations, cardiac and femoral blood, urine, gastric contents (40 g of reddishbrown ¯uid), bile, cerebrospinal ¯uid (CSF), vitreous humour (VH), liver and brain cortex were sampled. Plasma was prepared from femoral blood. 3. Toxicological analyses 3.1. Paraphernalia analyses The empty syringe was rinsed with methanol. After solvent evaporation at 37 8C under N2, the dried extract was dissolved in 100 m1 methanol and submitted to gaschromatography±mass spectrometry (GC±MS) analysis. Half of the sample was acetylated prior to GC±MS. The residual liquid of the Vetanarcol1 bottle was extracted according to our routine procedure under basic conditions and then submitted to the same analytical scheme. 3.2. Systematic drug screening A comprehensive screening [4] was performed on post mortem cardiac blood, urine and gastric contents using a combination of colour tests, immunoassays, and chromatographic techniques. Ethanol and volatile compounds in peripheral blood were analysed by head-space±gas-chromatography equipped with ¯ame ionisation detection (HS±GC±FID). Basic and neutral drugs in blood were screened after alkaline extraction by high-performance liquid-chromatography with diode-array (HPLC±DAD) and GC±MS. The blood extracts were acetylated prior to GC±MS analysis. The same extraction and GC±MS procedure were used for the analysis of gastric contents. Conjugated drugs and toxicants in urine were cleaved by acid hydrolysis before alkaline extraction. After derivatisation with acetic anhydride in pyridine, the extracts were screened by GC±MS. Unconjugated xenobiotics in urine were analysed straightforward by GC±MS after basic extraction [4]. 3.3. Quanti®cation of pentobarbital Pentobarbital concentrations were determined in biological ¯uids or tissues by GC±MS operating in the selected ion monitoring mode following acid extraction and ¯ash alkylation in the injector port of the GC with 0.2 M
trimethylanilinium hydroxide in methanol (TMAH, Supelco) [5,6]. Secobarbital was used as internal standard. The drug was extracted from 1 ml or 1 g of biological sample by mixing with 100 ml secobarbital (10 mg/ml), 2 ml of 0.5 M KH2PO4, pH 2:5 and 3 ml chloroform/isopropanol (9:1; v/v). After 10 min mixing on a horizontal shaker and centrifugation, the organic phase was collected and taken to dryness with N2 at 37 8C. Then, 100 ml of TMAH were added to the dry residue, and 1 ml was injected splitless into the GC±MS. To achieve full methylation of the barbiturates, the injector temperature was set at 250 8C. The oven temperature was held at 100 8C for 1 min and then increased in two steps ®rst to 200 8C at a rate of 20 8C/min and then to 300 8C at a rate of 15 8C/min. The latter temperature was kept constant for 8.3 min. Analysis was performed with an Agilent 6890 Series GC system interfaced with a HewlettPackard 5973 Mass Selective Detector using an HP-5MS capillary column (30 m 0:25 mm (i.d.) 0.25 mm). Helium was used as a carrier gas at a constant ¯ow-rate of 1.5 ml/min. The transfer line was maintained at a temperature of 280 8C. The following ions were recorded for the TMAH dimethylated barbiturate derivatives: pentobarbital, m/z 225, 184, 169; secobarbital, m/z 248, 196, 138. Calibration curves were constructed on 10 points ranging from 0.2 to 12 mg pentobarbital/ml of whole blood. The correlation coef®cient r2 was always higher than 0.995. 4. Results and discussion 4.1. Syringe and Vetanarcol1 analyses Although the extraction pH of our routine method was basic, pentobarbital was detected both in the rinsing solution of the syringe and in the 2 ml residual liquid left in the Vetanarcol1 bottle. 4.2. Toxicological ®ndings Barbiturates were detected in urine by radioimmunoassay and in blood by HPLC±DAD. All biological samples submitted to GC±MS analysis showed the presence of pentobarbital. The dehydrated form of its hydroxylated metabolite was detected in urine after acid hydrolysis and acetylation. The basic extract of urine revealed the presence of 30 -hydroxy-pentobarbital. Other components included nicotine, cotinine, caffeine and theobromine. Neither alcohol, nor other volatile compounds were detected in whole blood specimens. The biological ¯uid and tissue concentrations of pentobarbital are presented in Table 1. The peripheral blood (13.5 mg/ml) and plasma (21.7 mg/ ml) concentrations determined here are in the range of blood levels reported in cases of fatal pentobarbital overdose. They are also higher than the blood concentrations measured for suspected-drugged drivers (Table 2).
N. Romain et al. / Forensic Science International 131 (2003) 103±107 Table 1 Biological ¯uid and tissue concentrations of pentobarbital and estimation of total amount in gastric contents Specimen
Concentration (mg/ml or mg/g)
Peripheral blood 13.5 Plasma from peripheral blood 21.7 Plasma/blood ratio 1.6 Urine 7.2 Gastric contents 18.1 (0.7 mg total) Vitrous humor 12.6 Brain cortex 33.2 Cerebrospinal fluid 13.9 Bile 67.4 Liver 27.5 Vetanarcol1 162,000 10 ml syringe 162,000 (1620 mg total) Composition of the Vetanarcol1 bottle and amount of pentobarbital which could be contained in the empty syringe.
Assuming that the content of one 10 ml syringe was injected, one could calculate the total amount of pentobarbital which was administered; approximately 1.6 g. Taking into account this hypothesis, the peak plasma concentration could be estimated as follows: Cp 1620 mg (amount in body)/1 (volume of distribution) 69 (body weight) 23.5 mg/ml, a concentration which is very close to the plasma concentration determined in the present case (21.7 mg/ml) (Table 2). The peripheral plasma to whole blood ratio of 1.6 (Table 1) is higher than the plasma/whole blood ratio (0.96) reported in the literature [7]. A ratio close to 1 indicates that pentobarbital concentrations in blood red cells and in plasma are almost identical. Water losses from the vascular system and drug redistribution may account for the observed change in the distribution ratio value between plasma and whole blood compartments [11]. The low amount of pentobarbital recovered from gastric contents (0.7 mg) may be due to post mortem redistribution [18,21]. Indeed, an accumulative body of evidence (the presence of a syringe ®xed in the cap of the Vetanarcol1 bottle, the detection of Table 2 Pentobarbital: main pharmacological and toxicological data Parameter
Range
Reference
Therapeutic dose Minimum lethal dose Therapeutic blood level Toxic blood level Lethal blood concentrations Suspected-drugged drivers Plasma elimination half-life Plasma/whole blood ratio Volume of distribution Plasmatic protein binding
100±200 mg 1000 mg 1±3 mg/ml >5 mg/ml 10±169 mg/ml 0.1±4.9 mg/g 15±48 h 0.93 0.7±1.0 l/kg 65%
[7] [7] [8] [8] [8] [9] [10] [7] [7] [7]
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two punctured marks within an ecchymosis on the abdomen) suggests that pentobarbital was administered by injection. Suicides resulting from the ingestion of a high dose of a veterinarian preparation have also been reported in the literature [3,12]. It is interesting to note that the peripheral blood concentration, the cerebrospinal ¯uid level and the vitreous humour concentration were very similar. This suggests that these two latter samples could be used to estimate the blood concentration. It has been observed that vitreous drug concentrations often re¯ect circulating blood concentrations from 1 to 2 h prior to death [13,14]. Levels of pentobarbital in the CSF may re¯ect more closely than blood the active concentration of pentobarbital at its site of action, i.e. the central nervous system. Drugs which are bound to proteins will tend to have signi®cantly lower concentration in the CSF than in the blood. The partial binding of pentobarbital to plasma proteins (65%, Table 1) may explain why the plasma concentration is higher than the CSF level. Pentobarbital concentration in bile was very high compared to the urine level indicating that this body ¯uid could be used for the screening of barbiturates when no urine is available or when in spite of suspected overdose, because of rapid death, no barbiturate is detected in urine. This observation is in agreement with previous data that show that bile is a valuable matrix for the screening of a large array of xenobiotics and that bile concentrations are very often higher than those of other biological ¯uids [15]. Table 3 summarizes the toxicological ®ndings and the pentobarbital distribution reported up to now in the scienti®c literature. These data are compared to the results obtained for the present case. It is noteworthy that in several cases barbiturates were quantitated by UV spectrophotometry [3,12,17]and that no GC±MS comprehensive screenings and formal identi®cation were carried out [1,3,12,16±18]. In one case [1], no indication of the analytical method could be found. The ability of these rather unspeci®c methods [19,20] to discriminate several barbiturates and their metabolites remains questionable. In case [18], a systematic investigation of blood levels measured in samples taken from different vessels revealed that a 6-fold difference in concentration exists between the inferior vena cava (31.5 mg/ ml) and the right femoral vein (5.1 mg/ml). These large differences are very likely the consequence of drug diffusion and post mortem redistribution. To minimize these effects, it is generally recommended to quantify drugs in blood sampled from the femoral vein. This peripheral blood is thought to re¯ect more closely the ante mortem situation [21]. The blood levels reported up till now in the literature [1,5,12,16±18] as well as in this case ranged from 5.1 to 173 mg/ml. Because of their low speci®city, some results obtained through spectrophotometric methods could be overestimated. This exempli®es the need to gather more accurate data on pentobarbital levels in biological specimens following fatal overdose.
106
Table 3 Case comparison study of suicides by pentobarbital Clark and Jones [16]
Shaw [17]
Shaw [17]
Poklis and Hameli [12]
Pounder and Jones [18]
ReÂsieÁre et al. [3]
Giroud et al. [5]
This case
Preparation trade name
Socumb1
Lethal1
Unknown
Toxital1
Unknown
Dolethal1
Unknown
Vetanarcol1
Composition concentration or amount
Pentobarbital 360 mg/ml
Pentobarbital 400 mg/ml
Pentobarbital 178 mg/ml
Pentobarbital; amobarbital; secobarbital
Pentobarbital 18 g
Pentobarbital 10 g
Pentobarbital 162 mg/ml
Route of administration Occupation or circumstances
Intravenous Veterinary worker Unknown
Pentobarbital 260 mg/ml; amobarbital 130 mg/ml Intravenous Veterinary assistant GC±FID
Beuthanasia Special1 Pentobarbital 390 mg/ml
Oral Drug abuser
Oral Veterinarian
HPLC±UV
UV spectrophotometry
Oral Oral Suicide Veterinarian assistance ``Exit'' GC±MS GC±MS
Method of quantification Specimen Serum Blood Brain Liver Vitreous humor Urine Gastric contents Bile Heart CSF nd: not detected.
Intraperitoneal Oral Oral Veterinary University Veterinarian worker research assistant UV spectroUV spectroUV spectrophotometry photometry photometry Concentration in body fluids and tissues or total amount in gastric contents 30.7 mg/ml
45 mg/ml 48 mg/g 20 mg/g 20 mg/ml nd nd
173 mg/ml
113 mg/ml
150 mg/ml
112 mg/g 1580 mg/g
100 mg/g 458 mg/g
320 mg/ml
445 mg/ml 40 mg 3480 mg/g
8.3 mg/ml 12.1 mg 114 mg/ml 180 mg/g
2.1 g
Systemic blood: Barbiturates 5.1±31.5 mg/ml 369 mmol/l 22±33 mg/ml 89 mg/g 3.9 mg/ml 7.7 mg/ml 903 mg 26.7 mg/ml 21.5 mg/g
16.1±59.6 mg/ml
21.7 mg/g 13.5 mg/g 33.2 mg/g 27.5 mg/g 12.6 mg/g 7.2 mg/g 0.7 mg 67.4 mg/g 13.9 mg/g
N. Romain et al. / Forensic Science International 131 (2003) 103±107
Cordell et al. [1]
N. Romain et al. / Forensic Science International 131 (2003) 103±107
5. Conclusions When confronted with the death of a veterinary surgeon or of people working in a veterinary of®ce, overdose with euthanasia agent must be suspected and toxicological investigations should be carried out. Because veterinarians prefer to use very concentrated solutions of pentobarbital, preventing the risk of suicide attempt seems to be hardly possible. The use of small ampoules instead of large bottles and their storage in a secured place could however lower the risk.
[7] [8] [9] [10]
Acknowledgements
[11]
We are grateful to MS Anne Tricot for her excellent handling of this manuscript.
[12] [13]
References [1] W.H. Cordell, S.C. Curry, R.B. Furbee, D.L. Mitchell-Flynn, Veterinary euthanasia drugs as suicide agents, Ann. Emerg. Med. 15 (1986) 939±943. [2] M. Giorgi, S. Bertini, G. Soldani, M. Giusani, Comparison of HPLC and GC±MS methods for determination of embutramide (a compound of Tanax1 or T-611) in biological specimens, J. Anal. Toxicol. 25 (2001) 323±327. [3] D. ReÂsieÁre, B. MeÂgarbane, P. Manet, A. Buisine, F. Hilpert, Intoxication aigueÈ par le pentobarbital, Presse MeÂdicale 30 (2001) 269±270. [4] N. Romain, C. Giroud, K. Michaud, M. Augsburger, P. Mangin, Fatal ¯ecainide intoxication, Forensic Sci. Int. 106 (1999) 115±123. [5] C. Giroud, M. Augsburger, B. Horisberger, P. Lucchini, L. Rivier, P. Mangin, Exit association-mediated suicide: toxicologic and forensic aspects, Am. J. Forensic Med. Pathol. 20 (1999) 40±44. [6] P. Lillsunde, L. Michelson, T. ForsstroÈm, T. Korte, E. Schultz, M. Ariniemi, M. Portman, M.-L. Sihvonen, T. SeppaÈlaÈ, Comprehensive drug screening in blood for detecting abused
[14] [15] [16] [17] [18] [19]
[20] [21]
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drugs or drugs potentially hazardous for traf®c safety, Forensic Sci. Int. 77 (1996) 191±210. A.C. Moffat (Ed.), Clarke's Isolation and identi®cation of drugs in pharmaceuticals, body ¯uids, and post-mortem material, 2nd ed., The Pharmaceutical Press, London, 1986. C.L. Winek, W.W. Wahba, C.L. Winek, T.W. Balzer, Drug and chemical blood-level data 2001, Forensic Sci. Int. 122 (2001) 107±123. H. Druid, P. Holmgren, A compilation of fatal and control concentrations of drugs in postmortem femoral blood, J. Forensic Sci. 42 (1997) 79±87. M. Schulz, A. Schmoldt, Therapeutic and toxic blood concentrations of more than 500 drugs, Rev. Pharmazie 52 (1997) 895±911. R.W. Prouty, W.H. Anderson, The forensic implications of site and temporal differences on predicting blood-drug concentrations, J. Forensic Sci. 35 (1990) 243±270. A. Poklis, A.Z. Hameli, Two unusual barbiturate deaths, Arch. Toxicol. 34 (1975) 77±80. D.J. DiMaio, V.J.M. DiMaio, Forensic Pathology, CRC Press, Boca Raton, FL, 1993. J.L. Coe, Postmortem chemistry update: emphasis on forensic application, Am. J. Forensic Med. Pathol. 14 (1993) 91±117. A. Agarwal, N. Lemos, Signi®cance of bile analysis in druginduced deaths, J. Anal. Toxicol. 20 (1996) 61±63. M.A. Clark, J.W. Jones, Suicide by intravenous injection of veterinary euthanasia agent: report of a case and toxicologic studies, J. Forensic Sci. 23 (1979) 762±767. R.F. Shaw, Distribution of veterinarian barbiturate preparations in human subjects, Clin. Toxicol. 18 (1981) 965±968. D.J. Pounder, G.R. Jones, Post-mortem drug redistributionÐ a toxicological nightmare, Forensic Sci. Int. 45 (1990) 253± 263. P.M.G. Broughton, A rapid ultraviolet spectrophotometric method for the detection and estimation and identi®cation of barbiturates in biological material, Biochem. J. 63 (1956) 207±213. L.R. Goldbaum, Determination of barbiturates: ultraviolet spectrophotometric method with differentiation of several barbiturates, Anal. Chem. 24 (1952) 1604±1607. G.R. Jones, Interpretation of postmortem drug levels, in: S.B. Karch (Ed.), Drug Abuse Handbook, CRC Press, London, 1998, pp. 970±985.
Suicide by injection of a veterinarian barbiturate euthanasia agent: report of a case and toxicological analysis Nathalie Romain*, Christian Giroud, Katarzyna Michaud, Patrice Mangin Institut Universitaire de MeÂdecine LeÂgale de Lausanne, rue du Bugnon 21, 1005 Lausanne, Switzerland Received 10 July 2002; received in revised form 20 October 2002; accepted 6 November 2002
Abstract The history and toxicological ®ndings of a suicidal case involving injection of a veterinarian barbiturate euthanasia agent (Vetanarcol1) containing pentobarbital are presented. Blood pentobarbital concentrations compatible with drug overdose were determined. Almost identical levels were found in blood, cerebrospinal ¯uid (CSF) and vitreous humour (VH). The highest concentration was measured in the bile. The present case is compared with similar rare cases in the literature. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Suicide; Barbiturates; Pentobarbital; Euthanasia; Veterinary
1. Introduction Vetanarcol1 and T-611 are two pharmaceutical preparations very commonly used for the euthanasia of animals. Vetanarcol1 has been marketed in Switzerland since 1957 and is available in 100 ml bottle with each millilitre containing 162 mg of pentobarbital sodium-salt, 20 mg of benzyl alcohol as preservative and 500 mg of propylene glycol as vehicle. Depending on the dose, it is used by intravenous or intra-peritoneal administration for the animals narcosis or euthanasia. In the last case, it produces rapid central nervous system depression and cardiovascular collapse [1]. For instance, a dog is rapidly killed by injection of 0.5±1.0 ml/kg of Vetanarcol1. Another common procedure is to administrate Vetanarcol1 as a premedication before a lethal dose of T-611. T-611 is a mixture of three compounds containing embutramide, a general anaesthetic, mebenzonium, a muscle relaxant, and tetracaine, a local anaesthetic [2]. According to the Swiss law, pentobarbital is a controlled substance. Vetanarcol1 can be only bought by veterinary surgeons with a state licence for narcotics.
* Corresponding author. Tel.: 41-21-3147070; fax: 41-21-3147090. E-mail address: [email protected] (N. Romain).
On the one hand, accidental and volunteer overdoses caused by short-acting barbiturates in humans have become rare since these molecules became commercially unavailable as sleeping drugs/hypnotics [3]. On the other hand, suicidal ingestion of a lethal dose of pentobarbital has increased because its use is advocated by the ``Exit'' association. This association provides assistance to individuals wishing to commit suicide because of lasting painful disease or severe degradation of their physical condition. Infrequent suicides with pentobarbital in medical and paramedical profession, have also been reported. We report a fatal deliberate overdose attributed to Vetanarcol1. The toxicological ®ndings are presented and compared to those published in the literature. 2. Case history A 51-year-old man, administrator of the county society for the prevention of cruelty to animals was found dead in his apartment next to the refuge. At the scene of death, a 100 ml bottle of Vetanarcol1 containing about 2 ml of liquid, with an empty 10 ml syringe ®xed in its cap was found close to the body. Several farewell letters were discovered on the harddisk of his computer. The victim showed an abdominal ecchymosis with two punctured wounds. The autopsy was carried out 24 h after the discovery of the corpse and there was no feature of decomposition. The
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N. Romain et al. / Forensic Science International 131 (2003) 103±107
body weighed 69 kg. The right side of the abdomen showed a large (18 cm 10 cm) purple ecchymosis with two punctured wounds which could be considered as injection marks. No other traumatic lesion was noticed. Except a diffuse visceral congestion, autopsy ®ndings were unremarkable. Microscopic examination of organs showed no signi®cant pathologic changes. Suicidal administration of Vetanarcol1 was suspected as the cause of death. For toxicological investigations, cardiac and femoral blood, urine, gastric contents (40 g of reddishbrown ¯uid), bile, cerebrospinal ¯uid (CSF), vitreous humour (VH), liver and brain cortex were sampled. Plasma was prepared from femoral blood. 3. Toxicological analyses 3.1. Paraphernalia analyses The empty syringe was rinsed with methanol. After solvent evaporation at 37 8C under N2, the dried extract was dissolved in 100 m1 methanol and submitted to gaschromatography±mass spectrometry (GC±MS) analysis. Half of the sample was acetylated prior to GC±MS. The residual liquid of the Vetanarcol1 bottle was extracted according to our routine procedure under basic conditions and then submitted to the same analytical scheme. 3.2. Systematic drug screening A comprehensive screening [4] was performed on post mortem cardiac blood, urine and gastric contents using a combination of colour tests, immunoassays, and chromatographic techniques. Ethanol and volatile compounds in peripheral blood were analysed by head-space±gas-chromatography equipped with ¯ame ionisation detection (HS±GC±FID). Basic and neutral drugs in blood were screened after alkaline extraction by high-performance liquid-chromatography with diode-array (HPLC±DAD) and GC±MS. The blood extracts were acetylated prior to GC±MS analysis. The same extraction and GC±MS procedure were used for the analysis of gastric contents. Conjugated drugs and toxicants in urine were cleaved by acid hydrolysis before alkaline extraction. After derivatisation with acetic anhydride in pyridine, the extracts were screened by GC±MS. Unconjugated xenobiotics in urine were analysed straightforward by GC±MS after basic extraction [4]. 3.3. Quanti®cation of pentobarbital Pentobarbital concentrations were determined in biological ¯uids or tissues by GC±MS operating in the selected ion monitoring mode following acid extraction and ¯ash alkylation in the injector port of the GC with 0.2 M
trimethylanilinium hydroxide in methanol (TMAH, Supelco) [5,6]. Secobarbital was used as internal standard. The drug was extracted from 1 ml or 1 g of biological sample by mixing with 100 ml secobarbital (10 mg/ml), 2 ml of 0.5 M KH2PO4, pH 2:5 and 3 ml chloroform/isopropanol (9:1; v/v). After 10 min mixing on a horizontal shaker and centrifugation, the organic phase was collected and taken to dryness with N2 at 37 8C. Then, 100 ml of TMAH were added to the dry residue, and 1 ml was injected splitless into the GC±MS. To achieve full methylation of the barbiturates, the injector temperature was set at 250 8C. The oven temperature was held at 100 8C for 1 min and then increased in two steps ®rst to 200 8C at a rate of 20 8C/min and then to 300 8C at a rate of 15 8C/min. The latter temperature was kept constant for 8.3 min. Analysis was performed with an Agilent 6890 Series GC system interfaced with a HewlettPackard 5973 Mass Selective Detector using an HP-5MS capillary column (30 m 0:25 mm (i.d.) 0.25 mm). Helium was used as a carrier gas at a constant ¯ow-rate of 1.5 ml/min. The transfer line was maintained at a temperature of 280 8C. The following ions were recorded for the TMAH dimethylated barbiturate derivatives: pentobarbital, m/z 225, 184, 169; secobarbital, m/z 248, 196, 138. Calibration curves were constructed on 10 points ranging from 0.2 to 12 mg pentobarbital/ml of whole blood. The correlation coef®cient r2 was always higher than 0.995. 4. Results and discussion 4.1. Syringe and Vetanarcol1 analyses Although the extraction pH of our routine method was basic, pentobarbital was detected both in the rinsing solution of the syringe and in the 2 ml residual liquid left in the Vetanarcol1 bottle. 4.2. Toxicological ®ndings Barbiturates were detected in urine by radioimmunoassay and in blood by HPLC±DAD. All biological samples submitted to GC±MS analysis showed the presence of pentobarbital. The dehydrated form of its hydroxylated metabolite was detected in urine after acid hydrolysis and acetylation. The basic extract of urine revealed the presence of 30 -hydroxy-pentobarbital. Other components included nicotine, cotinine, caffeine and theobromine. Neither alcohol, nor other volatile compounds were detected in whole blood specimens. The biological ¯uid and tissue concentrations of pentobarbital are presented in Table 1. The peripheral blood (13.5 mg/ml) and plasma (21.7 mg/ ml) concentrations determined here are in the range of blood levels reported in cases of fatal pentobarbital overdose. They are also higher than the blood concentrations measured for suspected-drugged drivers (Table 2).
N. Romain et al. / Forensic Science International 131 (2003) 103±107 Table 1 Biological ¯uid and tissue concentrations of pentobarbital and estimation of total amount in gastric contents Specimen
Concentration (mg/ml or mg/g)
Peripheral blood 13.5 Plasma from peripheral blood 21.7 Plasma/blood ratio 1.6 Urine 7.2 Gastric contents 18.1 (0.7 mg total) Vitrous humor 12.6 Brain cortex 33.2 Cerebrospinal fluid 13.9 Bile 67.4 Liver 27.5 Vetanarcol1 162,000 10 ml syringe 162,000 (1620 mg total) Composition of the Vetanarcol1 bottle and amount of pentobarbital which could be contained in the empty syringe.
Assuming that the content of one 10 ml syringe was injected, one could calculate the total amount of pentobarbital which was administered; approximately 1.6 g. Taking into account this hypothesis, the peak plasma concentration could be estimated as follows: Cp 1620 mg (amount in body)/1 (volume of distribution) 69 (body weight) 23.5 mg/ml, a concentration which is very close to the plasma concentration determined in the present case (21.7 mg/ml) (Table 2). The peripheral plasma to whole blood ratio of 1.6 (Table 1) is higher than the plasma/whole blood ratio (0.96) reported in the literature [7]. A ratio close to 1 indicates that pentobarbital concentrations in blood red cells and in plasma are almost identical. Water losses from the vascular system and drug redistribution may account for the observed change in the distribution ratio value between plasma and whole blood compartments [11]. The low amount of pentobarbital recovered from gastric contents (0.7 mg) may be due to post mortem redistribution [18,21]. Indeed, an accumulative body of evidence (the presence of a syringe ®xed in the cap of the Vetanarcol1 bottle, the detection of Table 2 Pentobarbital: main pharmacological and toxicological data Parameter
Range
Reference
Therapeutic dose Minimum lethal dose Therapeutic blood level Toxic blood level Lethal blood concentrations Suspected-drugged drivers Plasma elimination half-life Plasma/whole blood ratio Volume of distribution Plasmatic protein binding
100±200 mg 1000 mg 1±3 mg/ml >5 mg/ml 10±169 mg/ml 0.1±4.9 mg/g 15±48 h 0.93 0.7±1.0 l/kg 65%
[7] [7] [8] [8] [8] [9] [10] [7] [7] [7]
105
two punctured marks within an ecchymosis on the abdomen) suggests that pentobarbital was administered by injection. Suicides resulting from the ingestion of a high dose of a veterinarian preparation have also been reported in the literature [3,12]. It is interesting to note that the peripheral blood concentration, the cerebrospinal ¯uid level and the vitreous humour concentration were very similar. This suggests that these two latter samples could be used to estimate the blood concentration. It has been observed that vitreous drug concentrations often re¯ect circulating blood concentrations from 1 to 2 h prior to death [13,14]. Levels of pentobarbital in the CSF may re¯ect more closely than blood the active concentration of pentobarbital at its site of action, i.e. the central nervous system. Drugs which are bound to proteins will tend to have signi®cantly lower concentration in the CSF than in the blood. The partial binding of pentobarbital to plasma proteins (65%, Table 1) may explain why the plasma concentration is higher than the CSF level. Pentobarbital concentration in bile was very high compared to the urine level indicating that this body ¯uid could be used for the screening of barbiturates when no urine is available or when in spite of suspected overdose, because of rapid death, no barbiturate is detected in urine. This observation is in agreement with previous data that show that bile is a valuable matrix for the screening of a large array of xenobiotics and that bile concentrations are very often higher than those of other biological ¯uids [15]. Table 3 summarizes the toxicological ®ndings and the pentobarbital distribution reported up to now in the scienti®c literature. These data are compared to the results obtained for the present case. It is noteworthy that in several cases barbiturates were quantitated by UV spectrophotometry [3,12,17]and that no GC±MS comprehensive screenings and formal identi®cation were carried out [1,3,12,16±18]. In one case [1], no indication of the analytical method could be found. The ability of these rather unspeci®c methods [19,20] to discriminate several barbiturates and their metabolites remains questionable. In case [18], a systematic investigation of blood levels measured in samples taken from different vessels revealed that a 6-fold difference in concentration exists between the inferior vena cava (31.5 mg/ ml) and the right femoral vein (5.1 mg/ml). These large differences are very likely the consequence of drug diffusion and post mortem redistribution. To minimize these effects, it is generally recommended to quantify drugs in blood sampled from the femoral vein. This peripheral blood is thought to re¯ect more closely the ante mortem situation [21]. The blood levels reported up till now in the literature [1,5,12,16±18] as well as in this case ranged from 5.1 to 173 mg/ml. Because of their low speci®city, some results obtained through spectrophotometric methods could be overestimated. This exempli®es the need to gather more accurate data on pentobarbital levels in biological specimens following fatal overdose.
106
Table 3 Case comparison study of suicides by pentobarbital Clark and Jones [16]
Shaw [17]
Shaw [17]
Poklis and Hameli [12]
Pounder and Jones [18]
ReÂsieÁre et al. [3]
Giroud et al. [5]
This case
Preparation trade name
Socumb1
Lethal1
Unknown
Toxital1
Unknown
Dolethal1
Unknown
Vetanarcol1
Composition concentration or amount
Pentobarbital 360 mg/ml
Pentobarbital 400 mg/ml
Pentobarbital 178 mg/ml
Pentobarbital; amobarbital; secobarbital
Pentobarbital 18 g
Pentobarbital 10 g
Pentobarbital 162 mg/ml
Route of administration Occupation or circumstances
Intravenous Veterinary worker Unknown
Pentobarbital 260 mg/ml; amobarbital 130 mg/ml Intravenous Veterinary assistant GC±FID
Beuthanasia Special1 Pentobarbital 390 mg/ml
Oral Drug abuser
Oral Veterinarian
HPLC±UV
UV spectrophotometry
Oral Oral Suicide Veterinarian assistance ``Exit'' GC±MS GC±MS
Method of quantification Specimen Serum Blood Brain Liver Vitreous humor Urine Gastric contents Bile Heart CSF nd: not detected.
Intraperitoneal Oral Oral Veterinary University Veterinarian worker research assistant UV spectroUV spectroUV spectrophotometry photometry photometry Concentration in body fluids and tissues or total amount in gastric contents 30.7 mg/ml
45 mg/ml 48 mg/g 20 mg/g 20 mg/ml nd nd
173 mg/ml
113 mg/ml
150 mg/ml
112 mg/g 1580 mg/g
100 mg/g 458 mg/g
320 mg/ml
445 mg/ml 40 mg 3480 mg/g
8.3 mg/ml 12.1 mg 114 mg/ml 180 mg/g
2.1 g
Systemic blood: Barbiturates 5.1±31.5 mg/ml 369 mmol/l 22±33 mg/ml 89 mg/g 3.9 mg/ml 7.7 mg/ml 903 mg 26.7 mg/ml 21.5 mg/g
16.1±59.6 mg/ml
21.7 mg/g 13.5 mg/g 33.2 mg/g 27.5 mg/g 12.6 mg/g 7.2 mg/g 0.7 mg 67.4 mg/g 13.9 mg/g
N. Romain et al. / Forensic Science International 131 (2003) 103±107
Cordell et al. [1]
N. Romain et al. / Forensic Science International 131 (2003) 103±107
5. Conclusions When confronted with the death of a veterinary surgeon or of people working in a veterinary of®ce, overdose with euthanasia agent must be suspected and toxicological investigations should be carried out. Because veterinarians prefer to use very concentrated solutions of pentobarbital, preventing the risk of suicide attempt seems to be hardly possible. The use of small ampoules instead of large bottles and their storage in a secured place could however lower the risk.
[7] [8] [9] [10]
Acknowledgements
[11]
We are grateful to MS Anne Tricot for her excellent handling of this manuscript.
[12] [13]
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