Buprenorphine Deaths in Singapore
Content
Forensic Science International 162 (2006) 80–86 www.elsevier.com/locate/forsciint
A survey of buprenorphine related deaths in Singapore
Siang Hui Lai a,*, Yi Ju Yao b, Danny Siaw Teck Lo b
a b
Centre for Forensic Medicine, Health Sciences Authority, Singapore, 11 Outram Road, Singapore 169078, Singapore Centre for Forensic Science, Health Sciences Authority, Singapore, 11 Outram Road, Singapore 169078, Singapore Received 26 August 2005; received in revised form 24 March 2006; accepted 24 March 2006 Available online 1 August 2006
Abstract Buprenorphine is available in Singapore as substitution treatment for opioid dependence since 2002. This study surveys buprenorphine related deaths in Singapore between September 2003 and December 2004. The aims are to establish the autopsy prevalence of buprenorphine related deaths and the demographical and toxicological profile of the cases. Toxicological screening was performed for all unnatural deaths, deaths involving known drug addicts, as well as when autopsy revealed no obvious cause of death. Twenty-one cases had buprenorphine detected in postmortem blood and/or urine samples. Eighteen were sudden deaths. There were two fatal falls from height and one death by hanging. All subjects were male. The age range was 24–48 years. Fourteen subjects were between 30 and 39 years of age. The mean age was 35 years. The majority (62%) were Chinese. Eleven (52%) were known drug abusers. For sudden deaths, two groups were identified. Six cases died from natural causes. Blood buprenorphine levels ranged from undetected (detected in urine) to 3.2 ng/mL (mean 1.4 ng/mL). Twelve cases were attributed directly and indirectly to mixed drug poisoning. Blood buprenorphine levels ranged from undetected (detected in urine) to 17 ng/mL (mean 3.2 ng/mL). Nineteen cases showed concurrent abuse of buprenorphine and benzodiazepine, diazepam being the most frequently detected, followed by nitrazepam and midazolam. The availability of buprenorphine as substitution therapy is associated with an increase in buprenorphine related deaths. The danger of co-abuse of buprenorphine and benzodiazepines is highlighted. # 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sudden deaths; Buprenorphine; Benzodiazepine; Opioid dependence
1. Introduction Buprenorphine has been available in Singapore as substitution therapy for opioid dependence since 2002. Sublingual preparations of buprenorphine in 2 and 8 mg tablets are available for substitution treatment. Currently, buprenorphine is not listed as a controlled drug in the Misuse of Drugs Act. There are also no specific regulations on the prescription of buprenorphine to opiate-dependent patients. The first case of buprenorphine related death in Singapore occurred in September 2003. This was a case of sudden death in a 35-year-old drug abuser. A hypodermic syringe found beside a deceased was analysed to contain buprenorphine. Coronial investigations ruled out other causes of death and death was attributed to mixed drug reaction due to buprenorphine and benzodiazepine.
Since then, with the increase awareness, the Centre for Forensic Medicine and Centre for Forensic Science, both of the Health Sciences Authority, have since recorded a sharp increase in the number of deaths associated with buprenorphine. This paper presents the data on buprenorphine related deaths in Singapore from the index case in September 2003 to December 2004 inclusive. The study period is from September 2003 to December 2004 inclusive. This retrospective survey aims: (a) To establish the autopsy prevalence and incidence of buprenorphine related deaths. (b) To establish the demographical data of deceased victims. (c) To identify particular patterns of drug abuse. 2. Methodology Coronial autopsies were performed on all sudden deaths, unnatural deaths and deaths under suspicious circumstances. Toxicological analyses on post-mortem samples of body fluids
* Corresponding author. Tel.: +65 62130651; fax: +65 62250217. E-mail address: [email protected] (S.H. Lai). 0379-0738/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2006.03.037
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were requested where indicated, for all cases of homicides, unnatural deaths, suspicious deaths and deaths of known or suspected drug abusers. Post-mortem blood and urine were subjected to full toxicological screen as described elsewhere [1]. In cases where there is no apparent cause of death, and/or benzodiazepines were detected in the biological specimens, or deceased is a known drug addict, the specimens were further screened for buprenorphine. From the autopsy and toxicology database, all cases with blood or urine samples positive for buprenorphine or its active metabolite norbuprenorphine within the study period were identified. All autopsy reports, including glass slides where available, and toxicology reports were retrieved for analysis. 2.1. Chemicals and reagents Buprenorphine hydrochloride (0.1 mg/mL in methanol) and norbuprenorphine hydrochloride (0.1 mg/mL in methanol) were purchased from Cerilliant (TX, USA). Internal standard clonazepam was obtained from Roche (Basel, Switzerland). All chemicals were of analytical-reagent grade and all solvents used were of HPLC grade.
Table 1 Calibration results and limits of detection and quantification Medium Blood Urine Analyte BUP NB BUP NB Concentration range (ng/mL) 0.25–12 1.25–60 0.5–32 2.5–160 Linear regression
2.2. Sample preparation Urine samples were screened for buprenorphine using enzyme-linked immunosorbent assay (ELISA) test kit from Neogen Corporation’s (KY, USA). A cut-off level of 1.0 ng/mL buprenorphine was employed. Urine samples screened positive for buprenorphine were extracted with 1-chlorobutane after basification with pH 12 sodium carbonate buffer, using clonazepam as the internal standard. Blood samples were similarly extracted and quantified for buprenorphine and norbuprenorphine [2]. 2.3. Chromatography An Agilent 1100 LC with VL quadrupole mass spectrometer was used for the quantification of buprenorphine and its metabolite norbuprenorphine. The chromatographic separation was obtained on a Hypersil-BDS column (150 mm  2.1 mm i.d., 5 mm particle size, Thermo Hypersil-Keystone, Cheshire, UK) using a gradient elution with mobile phase A consisting of aqueous 2 mM ammonium formate (pH 2.5 adjusted with 1% formic acid), and mobile phase B consisting of acetonitrile, at a flow rate of 0.4 mL/min. The initial condition of 10% B was
R2 0.9949 0.9965 0.9986 0.9933
LOD (ng/mL) 0.05 0.25 0.05 0.25
LOQ (ng/mL) 0.25 1.25 0.5 2.5
% CV at LOQ 7.6 7.8 14.3 10.5
Y = À0.0227 + 0.2479X Y = À0.0374 + 0.0553X Y = À0.0863 + 0.2646X Y = À0.0526 + 0.0406X
Table 2 Intra-day and inter-day precision and accuracy Analyte Spiked concentration (ng/mL) Concentration found (mean Æ S.D.) (ng/mL) 0.57 Æ 0.04 2.01 Æ 0.09 2.52 Æ 0.37 10.41 Æ 0.76 2.02 Æ 0.03 8.19 Æ 0.14 10.49 Æ 0.44 39.93 Æ 1.66 0.47 Æ 0.05 1.95 Æ 0.16 2.41 Æ 0.21 9.98 Æ 0.78 2.03 Æ 0.16 8.02 Æ 0.32 10.86 Æ 0.69 40.13 Æ 3.87 CV (%) Accuracy (%)
Intra-day precision and accuracy (n = 6) BUP in blood 0.5 2.0 NB in blood BUP in urine NB in urine 2.5 10.0 2.0 8.0 10.0 40.0
7.2 4.5 14.8 7.3 1.7 1.7 4.2 4.1 10.8 8.4 8.7 7.8 8.0 4.0 6.3 9.6
114.0 100.5 100.8 104.1 101.0 102.3 104.9 99.8 94.0 97.5 96.4 99.8 101.5 100.2 108.6 100.3
Inter-day precision and accuracy (n = 9) BUP in blood 0.5 2.0 NB in blood BUP in urine NB in urine 2.5 10.0 2.0 8.0 10.0 40.0
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S.H. Lai et al. / Forensic Science International 162 (2006) 80–86 Diazepam 0.74, nordiazepam 0.6, methorphan 0.16 Diazepam (traces), nordiazepam 0.03 Diazepam traces, nordiazepam 0.09, midazolam 0.29 Diazepam 0.69, nordiazepam 0.18, midazolam 0.02, zopiclone 0.68, orphenadrine 0.16 Not sampled ND ND ND Not sampled 0.6 0.7 1.1 ND ND ND D ND 1.3 0.4 1.6 Spontaneous rupture of diagonal branch of left coronary artery Coronary artery disease Bronchopneumonia Acute myocarditis
2.4. Validation Intra-day and inter-day precision and accuracy were determined on spiked samples at various concentrations. Precision is defined as the coefficient of variation (%), and accuracy is defined as a percentage of the target concentration. The limit of detection (LOD) is experimentally obtained, and is defined as the concentration that gives rise to a signal-tonoise ratio of 5. The limit of quantification (LOQ) is defined as the lowest level of analyte that can be accurately and precisely measured. Extraction recoveries of the analytes in blood and urine were determined by spiking blank matrices with the analytes at different concentrations. An additional set of blank matrices was also extracted with analytes added just before injection into the LC system. In all cases, the internal standard solution was added just before injection. The extraction recovery was obtained by comparing the relative peak analytes areas obtained in the two sets of samples. 3. Results
Urine norbuprenorphine level (ng/mL)
Urine buprenorphine level (ng/mL)
7.8
Blood norbuprenorphine level (ng/mL
Blood buprenorphine level (ng/mL)
3.2
Sepsis from disseminated abscesses (AIDS related)
Table 4 Group 1-sudden deaths with certified natural causes
Unemployed, complained of chest pain 1 month before Unemployed, known drug abuser, found in public toilet, syringe found in possession. Believed to have injected himself (injection mark identified) Unemployed, was foaming in the mouth, denied drug use 4 5 6 26/Ma 36/Ch 40/Ch
Food stall assistant, known drug abuser, Hep B and HIV positive, syringe found in possession, believed to have injected himself. Contractor, known drug abuser, undergoing drug rehabilitation Unemployed, no additional information
Validation of the LC/MS method for the determination of buprenorphine (BUP) and its metabolite norbuprenorphine (NB) in blood and urine samples is shown in Tables 1–3. The calibration curves, which were based on the peak area responses of the selected ions with respect to that of the internal standard clonazepam, were linear across the calibration range without special weighting or curve treatment.
Cause of death
Coronary artery disease ND—not detected; D—detected but not quantified.
3.1. LC/MS results
Table 3 Recovery of buprenorphine (BUP) and norbuprenorphine (NB) from blood and urine Medium Blood Analyte BUP NB Urine BUP NB Concentration range (ng/mL) 0.5 8 2.5 40 2 16 10 80 Mean Recovery (%) 87.4 84.5 100.6 88.0 82.7 91.9 63.2 73.5 Coefficient variation (%, n = 4) 12.2 3.4 6.5 4.9 1.5 4.7 10.9 6.0
Age/Race
Other information
31/Ma
Case
1
2
3
44/Ma
34/Ch
0.6
ND
D
2.5
1.7
D
Nitrazepam 0.03
Ketamine 0.03
Other drugs in blood (mcg/mL)
ramped to 65% B in the first 3 min, and hold at 65% for further 6 min. The following MS conditions were applied: electrospray ionisation (ESI) source, capillary voltage 4.5 kV, nebuliser pressure 45 psig, drying gas flow rate 13.0 L/min, drying gas temperature 350 8C, and fragmentor voltage 200 V. The monitoring ions were 316.1 (clonazepam I.S.), 468.3 (buprenorphine) and 414.3 (norbuprenorphine). Ions 469.3 and 415.3 were used as qualifier ions for buprenorphine and norbuprenorphine, respectively.
Table 5 Group 2-sudden deaths attributed to mixed drug reaction Case Age/Race Other information Blood buprenorphine level (ng/mL) 1 Blood norbuprenorphine level (ng/mL ND Urine buprenorphine level (ng/mL) 23 Urine norbuprenorphine level (ng/mL) ND Other drugs in blood (mcg/mL) S.H. Lai et al. / Forensic Science International 162 (2006) 80–86 Diazepam 0.19, nordiazepam 0.23, mirtazapine 0.07, ethanol 7 mg/dl Diazepam 0.03, midazolam 0.14, diphenhydramine 0.07, ethanol 6 mg/dl Diazepam 0.15, nordiazepam 0.14, nitrazepam (traces) Diazepam 0.55, nordiazepam 0.16, lignocaine 0.01 Ethanol 8 mg/dl
7
35/Ch
8 9 10 11
33/Ch 35/Ch 30/Ch 28/Ch
Self employed, known drug abuser, syringes found in possession, believed to have injected himself. Unemployed, known drug abuser Unemployed, known drug abuser, seen taking some white tablets 2 days before Unemployed, childhood asthma, pronounced dead at emergency department Cook, known drug abuser, syringe with white substance found in possession, believed to have injected himself. Port container lashing, known drug abuser, prescribed nitrados tablets Unemployed, no additional information Unemployed, drug abuser, undergoing drug rehabilitation Unemployed, known drug abuser, found collapsed at public place Unemployed, unconfirmed history of asthma Unemployed, no additional information Known drug abuser, found dead in a state of decomposition.
ND 0.7 5.7 2.5
ND ND ND ND
2 13 ND 3.4
ND ND 19.2 ND
12 13 14 15 16 17 18
31/Ind 31/Ma 24/Ch 36/Ch 37/Ma 32/Ch 37/Jav
0.8 0.8 17.1 4.4 0.8 0.8 1.1
ND ND 3.4 ND ND 0.8 ND
7.8 2.8 5.6 3.3 22.2 9.2 73.6
ND D 15.2 49.8 7.6 23 ND
Diazepam 0.21, nordiazepam 0.21, 7-aminonitrazepam (traces) Diazepam 0.37, nordiazepam 0.06 7-aminonitrazepam 0.28 Diazepam 0.22, nordiazepam 0.06, nitrazepam 0.32, diphenhydramine 0.62, 8-chlorotheophylline 8.9 Diazepam 0.35, nordiazepam 0.08, midazolam 0.01, 7-aminonitrazepam (traces) Midazolam 0.16, 7-aminonitrazepam (traces) Diazepam 0.05, nordiazepam 0.07, ethanol 28 mg/dl
ND—not detected; D—detected but not quantified.
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S.H. Lai et al. / Forensic Science International 162 (2006) 80–86 Toluene 12, diazepam 0.32, nordiazepam 1.1, midazolam 0.95, citalopram 0.35, benzhexol (traces) Midazolam 0.55 Not sampled a Not sampled a 2.4 Multiple injuries D ND D 68.6 26.9 Multiple injuries Nitrazepam 0.09, 7-aminonitrazepam (traces) Hanging ND—not detected; D—detected but not quantified. a Bile contained similar drug profile, vitreous humour negative. 21 48/Jav 2.6 20.5 8.3 359
3.2. Autopsy results In 2003, there were 3514 cases reported and investigated by the State Coroner. Of these, 1942 cases were subjected to autopsies. The figures for 2004 were 3422 and 1885, respectively. A total of 21 cases of buprenorphine related deaths were identified. There were two cases in the last 4 months of 2003. For the whole year of 2004, there were 19 cases. The incidence of buprenorphine related deaths was 1.0% in 2004. All victims were male. Eleven had a history of drug abuse. The others had unspecified drug abuse history. Only five had known occupations. The racial distribution of the victims was as follows: Chinese, 13 (61%); Malay, 5 (24%); Javanese, 2 (10%) and Indian, 1 (5%). The age distribution of the victims was as follows: less than 25 years old, 1 case; 25–29 years, 2 cases; 30– 34 years, 8 cases; 35–39 years, 6 cases; 40–44 years, 2 cases and 45–49 years, 2 cases. The age range of the victims was from 24 to 48 years. Two-thirds of the victims were between 30 and 39 years of age. The mean age was 34.7 years. The cases were classified into three categories as defined by the circumstances and certified causes of death. There were 18 cases of sudden deaths and three cases of deaths from trauma and other causes. The groupings were as follows: Group 1— deaths due to natural causes, Group 2—deaths attributed to buprenorphine, and Group 3—deaths due to other unnatural causes. Six cases of sudden deaths, Group 1 (Table 4), revealed sufficient anatomical pathology for death to be ascribed. Half of the deceased were Chinese. The mean age was 35.2 years. Review of the autopsy findings confirmed certified diagnoses. It is interesting to note that two cases (Cases 1 and 5), both known drug abusers were also believed to have injected themselves before they died. The lungs of these two cases showed typical features of chronic intravenous drug abuse. An injection mark was even observed at autopsy for Case 5. Unfortunately, the skin at the site was not submitted for toxicological and microscopic analysis. In one case (Case 4), buprenorphine was not detected in the post-mortem blood sample but present in the urine. The range of buprenorphine in the blood was 0.4 to 3.2 ng/mL. There were other 12 cases of sudden deaths in Group 2 (Table 5). Eight were Chinese. The mean age was 32.4 years. Case 18 was a case of decomposed corpse where autopsy failed to reveal any anatomical pathological cause of death. It was included in this group for although the cause of death was certified as ‘Unascertained’, the circumstances suggested that death was most likely attributable to the effects of mixed drug reaction. Seven were known drug abusers. Two were found dead with syringes in their possession and at least another was witnessed to have consumed drugs before their death. Review of histology slides confirmed features of chronic intravenous drug abuse in six cases. The index case (Case 7) of this entire series was believed to have injected himself with the drugs through analyses of the syringes present around the body. In one case
Cause of death
Blood buprenorphine level (ng/mL) Table 6 Group 3 cases of deaths due to other unnatural causes Age/Race Other information
Blood norbuprenorphine level (ng/mL
Urine buprenorphine level (ng/mL)
Urine norbuprenorphine level (ng/mL)
Other drugs in blood (mcg/mL)
33/Ch
Case
19
20
47/Ch
Unemployed, fell from 10 storeys whilst trying to hang bird cage outside flat, syringe and packets of yellow substance found in possession Unemployed, fell from 11 storeys, suicidal intentions. Unemployed, known drug abuser, hanged himself at home
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(Case 8), none was detected in the blood but detected in the urine sample. The range of buprenorphine detected in postmortem blood was 0.8–17.1 ng/mL. The level of buprenorphine detected in the post-mortem blood of Case 13 was above the reported therapeutic range of 1–8 ng/mL, as quoted for opiatedependent subjects undergoing 8 mg sublingual buprenorphine per day maintenance program [3]. Group 3 (Table 6) lists cases that were unnatural deaths not attributable to drugs. Case 19 was quite clearly a case of multidrug abuse. The combined effects of the drugs would have significantly altered the victim’s conscious level and his ability to co-ordinate his motor functions. 4. Discussion Sublingual buprenorphine has been reported to be as effective and safer than oral methadone in the treatment of opioid dependence. It has been approved in the United States of America for prescription in the office setting for this purpose under the Drug Addiction Treatment Act (DATA) of 2000 [4]. In France, sublingual buprenorphine was available as substitution therapy since 1996. Traqui et al. reported a series of 20 fatalities associated with the use of buprenorphine. Three risk factors were highlighted: diversion of sublingual buprenorphine tablets for intravenous use, the high dosage formulation available, and the concurrent abuse of benzodiazepine [5]. As long ago as 1979, Downing et al. reported that intravenous administration of buprenorphine could cause reduction in mean respiratory rate and increase in mean arterial carbon dioxide tension [6]. The depressive effect of buprenorphine on respiration was dose-dependent [7]. The product information for buprenorphine (Buprenex1) stated that respiratory and cardiovascular collapse have occurred in patients receiving therapeutic doses of diazepam and buprenorphine [8]. Animal studies confirmed the potential dangers of the concurrent administration of buprenorphine and benzodiazepines [9,10]. The findings in the current series further emphasised the possible dangers of diverted intravenous abuse of buprenorphine. The danger was apparent even where the post-mortem blood levels were found to be within the therapeutic range [3]. It underscored the danger of concurrent administration of buprenorphine with benzodiazepines. The mechanism of interaction between buprenorphine and benzodiazepines appeared not to be dose-dependent. Current data suggested that buprenorphine could be life-threatening without overdosage when concurrently used with other psychotropic drugs [5,11]. The small sample size of the present study failed to demonstrate any cut-off for the post-mortem blood buprenorphine level for which death could be attributed to mixed drug reaction. Certification of the cause of death after autopsy therefore depended on balancing the significance of anatomical pathology and toxicological findings and becomes a matter of opinion. On the balance of probability, it was usually more defensible and sensible to attribute cause of death to evident pathology rather than evoke reasonable doubt.
There were several limitations in this study, mainly inherent in coronial casework. Firstly, coronial casework dealt with a skewed sample population, which did not necessarily reflect the general population. Secondly, there was already a selection bias in which cases were subjected to autopsies. Coronial cases that subsequently revealed significant medical history would not be autopsied unless the circumstances indicate otherwise. Requests for toxicological analyses also depended greatly on circumstances of death and autopsy findings. Similarly, toxicological screenings were also carried out in stages, based on the same variables. Thirdly, the sample size for this study was small, hence limiting the power to discern statistical significance in any observed differences. 5. Conclusion In summary, buprenorphine has been approved as substitution therapy for opioid dependence in Singapore. There was a sharp increase in the incidence of buprenorphine related deaths in the first 2 years since its availability in 2002. All fatalities in this period involved males, many who were known drug abusers. In addition, most were Chinese and under the age of 40 years. This study highlighted that concurrent administration of buprenorphine and benzodiazepines was potentially fatal. The diversion of sublingual buprenorphine preparations for intravenous use presented an additional danger. The medical community must be alerted to these potential dangers. The relevant authorities and government agencies must also act promptly to regulate the prescription of buprenorphine, in order to prevent further fatalities from the misuse of buprenorphine. Acknowledgements The authors thank the State Coroner of Singapore for permission to report these cases. The main author wishes to thank colleagues at the Centre for Forensic Medicine for permission to review their cases. References
[1] Y.J. Yao, T.L. Ng, D.S.T. Lo, P.C.T. Eng, New approach to emergency toxicology—a case study, TIAFT Bull. 30 (4) (2000) 6–7. [2] Y.J. Yao, D.S.T. Lo, J.Y. Sim, T.H. Koh, Analysis of buprenorphine and norbuprenorphine in blood and urine by liquid chromatography-electrospray-mass spectrometry (LC-ES-MS), in: Presented in the 43rd The International Association of Forensic Toxicologists Meeting, 2005. [3] J.P. de los Cobos, S. Martin, A. Etcheberrigaray, J. Trujols, F. Batlle, A. Tejero, et al., A controlled trial of daily versus thrice-weekly buprenorphine administration for the treatment of opioid dependence, Drug Alcohol Depend. 59 (3) (2000) 223–233. [4] FDA Talk Paper T02-38 October 8, 2002, http://www.fda.gov/bbs/topics/ ANSWERS/2002/ANS01165.html, accessed on 11 July 2005. [5] A. Traqui, P. Kintz, B. Ludes, Buprenorphine-related deaths among drug addicts in France: a report on 20 fatalities, J. Anal. Toxicol. 22 (6) (1998) 430–434.
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[6] J.W. Downing, N.M. Goodwin, J. Hicks, The respiratory depressive effects of intravenous buprenorphine in patients in an intensive care unit, S. Afr. Med. J. 55 (25) (1979) 1023–1027. [7] R.C. Heel, R.N. Brogden, T.M. Speight, G.S. Avery, Buprenorphine: a review of its pharmacological properties and therapeutic efficacy, Drugs 17 (2) (1979) 81–110. [8] Buprenex1, Product Info 2001.
A survey of buprenorphine related deaths in Singapore
Siang Hui Lai a,*, Yi Ju Yao b, Danny Siaw Teck Lo b
a b
Centre for Forensic Medicine, Health Sciences Authority, Singapore, 11 Outram Road, Singapore 169078, Singapore Centre for Forensic Science, Health Sciences Authority, Singapore, 11 Outram Road, Singapore 169078, Singapore Received 26 August 2005; received in revised form 24 March 2006; accepted 24 March 2006 Available online 1 August 2006
Abstract Buprenorphine is available in Singapore as substitution treatment for opioid dependence since 2002. This study surveys buprenorphine related deaths in Singapore between September 2003 and December 2004. The aims are to establish the autopsy prevalence of buprenorphine related deaths and the demographical and toxicological profile of the cases. Toxicological screening was performed for all unnatural deaths, deaths involving known drug addicts, as well as when autopsy revealed no obvious cause of death. Twenty-one cases had buprenorphine detected in postmortem blood and/or urine samples. Eighteen were sudden deaths. There were two fatal falls from height and one death by hanging. All subjects were male. The age range was 24–48 years. Fourteen subjects were between 30 and 39 years of age. The mean age was 35 years. The majority (62%) were Chinese. Eleven (52%) were known drug abusers. For sudden deaths, two groups were identified. Six cases died from natural causes. Blood buprenorphine levels ranged from undetected (detected in urine) to 3.2 ng/mL (mean 1.4 ng/mL). Twelve cases were attributed directly and indirectly to mixed drug poisoning. Blood buprenorphine levels ranged from undetected (detected in urine) to 17 ng/mL (mean 3.2 ng/mL). Nineteen cases showed concurrent abuse of buprenorphine and benzodiazepine, diazepam being the most frequently detected, followed by nitrazepam and midazolam. The availability of buprenorphine as substitution therapy is associated with an increase in buprenorphine related deaths. The danger of co-abuse of buprenorphine and benzodiazepines is highlighted. # 2006 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sudden deaths; Buprenorphine; Benzodiazepine; Opioid dependence
1. Introduction Buprenorphine has been available in Singapore as substitution therapy for opioid dependence since 2002. Sublingual preparations of buprenorphine in 2 and 8 mg tablets are available for substitution treatment. Currently, buprenorphine is not listed as a controlled drug in the Misuse of Drugs Act. There are also no specific regulations on the prescription of buprenorphine to opiate-dependent patients. The first case of buprenorphine related death in Singapore occurred in September 2003. This was a case of sudden death in a 35-year-old drug abuser. A hypodermic syringe found beside a deceased was analysed to contain buprenorphine. Coronial investigations ruled out other causes of death and death was attributed to mixed drug reaction due to buprenorphine and benzodiazepine.
Since then, with the increase awareness, the Centre for Forensic Medicine and Centre for Forensic Science, both of the Health Sciences Authority, have since recorded a sharp increase in the number of deaths associated with buprenorphine. This paper presents the data on buprenorphine related deaths in Singapore from the index case in September 2003 to December 2004 inclusive. The study period is from September 2003 to December 2004 inclusive. This retrospective survey aims: (a) To establish the autopsy prevalence and incidence of buprenorphine related deaths. (b) To establish the demographical data of deceased victims. (c) To identify particular patterns of drug abuse. 2. Methodology Coronial autopsies were performed on all sudden deaths, unnatural deaths and deaths under suspicious circumstances. Toxicological analyses on post-mortem samples of body fluids
* Corresponding author. Tel.: +65 62130651; fax: +65 62250217. E-mail address: [email protected] (S.H. Lai). 0379-0738/$ – see front matter # 2006 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2006.03.037
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were requested where indicated, for all cases of homicides, unnatural deaths, suspicious deaths and deaths of known or suspected drug abusers. Post-mortem blood and urine were subjected to full toxicological screen as described elsewhere [1]. In cases where there is no apparent cause of death, and/or benzodiazepines were detected in the biological specimens, or deceased is a known drug addict, the specimens were further screened for buprenorphine. From the autopsy and toxicology database, all cases with blood or urine samples positive for buprenorphine or its active metabolite norbuprenorphine within the study period were identified. All autopsy reports, including glass slides where available, and toxicology reports were retrieved for analysis. 2.1. Chemicals and reagents Buprenorphine hydrochloride (0.1 mg/mL in methanol) and norbuprenorphine hydrochloride (0.1 mg/mL in methanol) were purchased from Cerilliant (TX, USA). Internal standard clonazepam was obtained from Roche (Basel, Switzerland). All chemicals were of analytical-reagent grade and all solvents used were of HPLC grade.
Table 1 Calibration results and limits of detection and quantification Medium Blood Urine Analyte BUP NB BUP NB Concentration range (ng/mL) 0.25–12 1.25–60 0.5–32 2.5–160 Linear regression
2.2. Sample preparation Urine samples were screened for buprenorphine using enzyme-linked immunosorbent assay (ELISA) test kit from Neogen Corporation’s (KY, USA). A cut-off level of 1.0 ng/mL buprenorphine was employed. Urine samples screened positive for buprenorphine were extracted with 1-chlorobutane after basification with pH 12 sodium carbonate buffer, using clonazepam as the internal standard. Blood samples were similarly extracted and quantified for buprenorphine and norbuprenorphine [2]. 2.3. Chromatography An Agilent 1100 LC with VL quadrupole mass spectrometer was used for the quantification of buprenorphine and its metabolite norbuprenorphine. The chromatographic separation was obtained on a Hypersil-BDS column (150 mm  2.1 mm i.d., 5 mm particle size, Thermo Hypersil-Keystone, Cheshire, UK) using a gradient elution with mobile phase A consisting of aqueous 2 mM ammonium formate (pH 2.5 adjusted with 1% formic acid), and mobile phase B consisting of acetonitrile, at a flow rate of 0.4 mL/min. The initial condition of 10% B was
R2 0.9949 0.9965 0.9986 0.9933
LOD (ng/mL) 0.05 0.25 0.05 0.25
LOQ (ng/mL) 0.25 1.25 0.5 2.5
% CV at LOQ 7.6 7.8 14.3 10.5
Y = À0.0227 + 0.2479X Y = À0.0374 + 0.0553X Y = À0.0863 + 0.2646X Y = À0.0526 + 0.0406X
Table 2 Intra-day and inter-day precision and accuracy Analyte Spiked concentration (ng/mL) Concentration found (mean Æ S.D.) (ng/mL) 0.57 Æ 0.04 2.01 Æ 0.09 2.52 Æ 0.37 10.41 Æ 0.76 2.02 Æ 0.03 8.19 Æ 0.14 10.49 Æ 0.44 39.93 Æ 1.66 0.47 Æ 0.05 1.95 Æ 0.16 2.41 Æ 0.21 9.98 Æ 0.78 2.03 Æ 0.16 8.02 Æ 0.32 10.86 Æ 0.69 40.13 Æ 3.87 CV (%) Accuracy (%)
Intra-day precision and accuracy (n = 6) BUP in blood 0.5 2.0 NB in blood BUP in urine NB in urine 2.5 10.0 2.0 8.0 10.0 40.0
7.2 4.5 14.8 7.3 1.7 1.7 4.2 4.1 10.8 8.4 8.7 7.8 8.0 4.0 6.3 9.6
114.0 100.5 100.8 104.1 101.0 102.3 104.9 99.8 94.0 97.5 96.4 99.8 101.5 100.2 108.6 100.3
Inter-day precision and accuracy (n = 9) BUP in blood 0.5 2.0 NB in blood BUP in urine NB in urine 2.5 10.0 2.0 8.0 10.0 40.0
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S.H. Lai et al. / Forensic Science International 162 (2006) 80–86 Diazepam 0.74, nordiazepam 0.6, methorphan 0.16 Diazepam (traces), nordiazepam 0.03 Diazepam traces, nordiazepam 0.09, midazolam 0.29 Diazepam 0.69, nordiazepam 0.18, midazolam 0.02, zopiclone 0.68, orphenadrine 0.16 Not sampled ND ND ND Not sampled 0.6 0.7 1.1 ND ND ND D ND 1.3 0.4 1.6 Spontaneous rupture of diagonal branch of left coronary artery Coronary artery disease Bronchopneumonia Acute myocarditis
2.4. Validation Intra-day and inter-day precision and accuracy were determined on spiked samples at various concentrations. Precision is defined as the coefficient of variation (%), and accuracy is defined as a percentage of the target concentration. The limit of detection (LOD) is experimentally obtained, and is defined as the concentration that gives rise to a signal-tonoise ratio of 5. The limit of quantification (LOQ) is defined as the lowest level of analyte that can be accurately and precisely measured. Extraction recoveries of the analytes in blood and urine were determined by spiking blank matrices with the analytes at different concentrations. An additional set of blank matrices was also extracted with analytes added just before injection into the LC system. In all cases, the internal standard solution was added just before injection. The extraction recovery was obtained by comparing the relative peak analytes areas obtained in the two sets of samples. 3. Results
Urine norbuprenorphine level (ng/mL)
Urine buprenorphine level (ng/mL)
7.8
Blood norbuprenorphine level (ng/mL
Blood buprenorphine level (ng/mL)
3.2
Sepsis from disseminated abscesses (AIDS related)
Table 4 Group 1-sudden deaths with certified natural causes
Unemployed, complained of chest pain 1 month before Unemployed, known drug abuser, found in public toilet, syringe found in possession. Believed to have injected himself (injection mark identified) Unemployed, was foaming in the mouth, denied drug use 4 5 6 26/Ma 36/Ch 40/Ch
Food stall assistant, known drug abuser, Hep B and HIV positive, syringe found in possession, believed to have injected himself. Contractor, known drug abuser, undergoing drug rehabilitation Unemployed, no additional information
Validation of the LC/MS method for the determination of buprenorphine (BUP) and its metabolite norbuprenorphine (NB) in blood and urine samples is shown in Tables 1–3. The calibration curves, which were based on the peak area responses of the selected ions with respect to that of the internal standard clonazepam, were linear across the calibration range without special weighting or curve treatment.
Cause of death
Coronary artery disease ND—not detected; D—detected but not quantified.
3.1. LC/MS results
Table 3 Recovery of buprenorphine (BUP) and norbuprenorphine (NB) from blood and urine Medium Blood Analyte BUP NB Urine BUP NB Concentration range (ng/mL) 0.5 8 2.5 40 2 16 10 80 Mean Recovery (%) 87.4 84.5 100.6 88.0 82.7 91.9 63.2 73.5 Coefficient variation (%, n = 4) 12.2 3.4 6.5 4.9 1.5 4.7 10.9 6.0
Age/Race
Other information
31/Ma
Case
1
2
3
44/Ma
34/Ch
0.6
ND
D
2.5
1.7
D
Nitrazepam 0.03
Ketamine 0.03
Other drugs in blood (mcg/mL)
ramped to 65% B in the first 3 min, and hold at 65% for further 6 min. The following MS conditions were applied: electrospray ionisation (ESI) source, capillary voltage 4.5 kV, nebuliser pressure 45 psig, drying gas flow rate 13.0 L/min, drying gas temperature 350 8C, and fragmentor voltage 200 V. The monitoring ions were 316.1 (clonazepam I.S.), 468.3 (buprenorphine) and 414.3 (norbuprenorphine). Ions 469.3 and 415.3 were used as qualifier ions for buprenorphine and norbuprenorphine, respectively.
Table 5 Group 2-sudden deaths attributed to mixed drug reaction Case Age/Race Other information Blood buprenorphine level (ng/mL) 1 Blood norbuprenorphine level (ng/mL ND Urine buprenorphine level (ng/mL) 23 Urine norbuprenorphine level (ng/mL) ND Other drugs in blood (mcg/mL) S.H. Lai et al. / Forensic Science International 162 (2006) 80–86 Diazepam 0.19, nordiazepam 0.23, mirtazapine 0.07, ethanol 7 mg/dl Diazepam 0.03, midazolam 0.14, diphenhydramine 0.07, ethanol 6 mg/dl Diazepam 0.15, nordiazepam 0.14, nitrazepam (traces) Diazepam 0.55, nordiazepam 0.16, lignocaine 0.01 Ethanol 8 mg/dl
7
35/Ch
8 9 10 11
33/Ch 35/Ch 30/Ch 28/Ch
Self employed, known drug abuser, syringes found in possession, believed to have injected himself. Unemployed, known drug abuser Unemployed, known drug abuser, seen taking some white tablets 2 days before Unemployed, childhood asthma, pronounced dead at emergency department Cook, known drug abuser, syringe with white substance found in possession, believed to have injected himself. Port container lashing, known drug abuser, prescribed nitrados tablets Unemployed, no additional information Unemployed, drug abuser, undergoing drug rehabilitation Unemployed, known drug abuser, found collapsed at public place Unemployed, unconfirmed history of asthma Unemployed, no additional information Known drug abuser, found dead in a state of decomposition.
ND 0.7 5.7 2.5
ND ND ND ND
2 13 ND 3.4
ND ND 19.2 ND
12 13 14 15 16 17 18
31/Ind 31/Ma 24/Ch 36/Ch 37/Ma 32/Ch 37/Jav
0.8 0.8 17.1 4.4 0.8 0.8 1.1
ND ND 3.4 ND ND 0.8 ND
7.8 2.8 5.6 3.3 22.2 9.2 73.6
ND D 15.2 49.8 7.6 23 ND
Diazepam 0.21, nordiazepam 0.21, 7-aminonitrazepam (traces) Diazepam 0.37, nordiazepam 0.06 7-aminonitrazepam 0.28 Diazepam 0.22, nordiazepam 0.06, nitrazepam 0.32, diphenhydramine 0.62, 8-chlorotheophylline 8.9 Diazepam 0.35, nordiazepam 0.08, midazolam 0.01, 7-aminonitrazepam (traces) Midazolam 0.16, 7-aminonitrazepam (traces) Diazepam 0.05, nordiazepam 0.07, ethanol 28 mg/dl
ND—not detected; D—detected but not quantified.
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S.H. Lai et al. / Forensic Science International 162 (2006) 80–86 Toluene 12, diazepam 0.32, nordiazepam 1.1, midazolam 0.95, citalopram 0.35, benzhexol (traces) Midazolam 0.55 Not sampled a Not sampled a 2.4 Multiple injuries D ND D 68.6 26.9 Multiple injuries Nitrazepam 0.09, 7-aminonitrazepam (traces) Hanging ND—not detected; D—detected but not quantified. a Bile contained similar drug profile, vitreous humour negative. 21 48/Jav 2.6 20.5 8.3 359
3.2. Autopsy results In 2003, there were 3514 cases reported and investigated by the State Coroner. Of these, 1942 cases were subjected to autopsies. The figures for 2004 were 3422 and 1885, respectively. A total of 21 cases of buprenorphine related deaths were identified. There were two cases in the last 4 months of 2003. For the whole year of 2004, there were 19 cases. The incidence of buprenorphine related deaths was 1.0% in 2004. All victims were male. Eleven had a history of drug abuse. The others had unspecified drug abuse history. Only five had known occupations. The racial distribution of the victims was as follows: Chinese, 13 (61%); Malay, 5 (24%); Javanese, 2 (10%) and Indian, 1 (5%). The age distribution of the victims was as follows: less than 25 years old, 1 case; 25–29 years, 2 cases; 30– 34 years, 8 cases; 35–39 years, 6 cases; 40–44 years, 2 cases and 45–49 years, 2 cases. The age range of the victims was from 24 to 48 years. Two-thirds of the victims were between 30 and 39 years of age. The mean age was 34.7 years. The cases were classified into three categories as defined by the circumstances and certified causes of death. There were 18 cases of sudden deaths and three cases of deaths from trauma and other causes. The groupings were as follows: Group 1— deaths due to natural causes, Group 2—deaths attributed to buprenorphine, and Group 3—deaths due to other unnatural causes. Six cases of sudden deaths, Group 1 (Table 4), revealed sufficient anatomical pathology for death to be ascribed. Half of the deceased were Chinese. The mean age was 35.2 years. Review of the autopsy findings confirmed certified diagnoses. It is interesting to note that two cases (Cases 1 and 5), both known drug abusers were also believed to have injected themselves before they died. The lungs of these two cases showed typical features of chronic intravenous drug abuse. An injection mark was even observed at autopsy for Case 5. Unfortunately, the skin at the site was not submitted for toxicological and microscopic analysis. In one case (Case 4), buprenorphine was not detected in the post-mortem blood sample but present in the urine. The range of buprenorphine in the blood was 0.4 to 3.2 ng/mL. There were other 12 cases of sudden deaths in Group 2 (Table 5). Eight were Chinese. The mean age was 32.4 years. Case 18 was a case of decomposed corpse where autopsy failed to reveal any anatomical pathological cause of death. It was included in this group for although the cause of death was certified as ‘Unascertained’, the circumstances suggested that death was most likely attributable to the effects of mixed drug reaction. Seven were known drug abusers. Two were found dead with syringes in their possession and at least another was witnessed to have consumed drugs before their death. Review of histology slides confirmed features of chronic intravenous drug abuse in six cases. The index case (Case 7) of this entire series was believed to have injected himself with the drugs through analyses of the syringes present around the body. In one case
Cause of death
Blood buprenorphine level (ng/mL) Table 6 Group 3 cases of deaths due to other unnatural causes Age/Race Other information
Blood norbuprenorphine level (ng/mL
Urine buprenorphine level (ng/mL)
Urine norbuprenorphine level (ng/mL)
Other drugs in blood (mcg/mL)
33/Ch
Case
19
20
47/Ch
Unemployed, fell from 10 storeys whilst trying to hang bird cage outside flat, syringe and packets of yellow substance found in possession Unemployed, fell from 11 storeys, suicidal intentions. Unemployed, known drug abuser, hanged himself at home
S.H. Lai et al. / Forensic Science International 162 (2006) 80–86
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(Case 8), none was detected in the blood but detected in the urine sample. The range of buprenorphine detected in postmortem blood was 0.8–17.1 ng/mL. The level of buprenorphine detected in the post-mortem blood of Case 13 was above the reported therapeutic range of 1–8 ng/mL, as quoted for opiatedependent subjects undergoing 8 mg sublingual buprenorphine per day maintenance program [3]. Group 3 (Table 6) lists cases that were unnatural deaths not attributable to drugs. Case 19 was quite clearly a case of multidrug abuse. The combined effects of the drugs would have significantly altered the victim’s conscious level and his ability to co-ordinate his motor functions. 4. Discussion Sublingual buprenorphine has been reported to be as effective and safer than oral methadone in the treatment of opioid dependence. It has been approved in the United States of America for prescription in the office setting for this purpose under the Drug Addiction Treatment Act (DATA) of 2000 [4]. In France, sublingual buprenorphine was available as substitution therapy since 1996. Traqui et al. reported a series of 20 fatalities associated with the use of buprenorphine. Three risk factors were highlighted: diversion of sublingual buprenorphine tablets for intravenous use, the high dosage formulation available, and the concurrent abuse of benzodiazepine [5]. As long ago as 1979, Downing et al. reported that intravenous administration of buprenorphine could cause reduction in mean respiratory rate and increase in mean arterial carbon dioxide tension [6]. The depressive effect of buprenorphine on respiration was dose-dependent [7]. The product information for buprenorphine (Buprenex1) stated that respiratory and cardiovascular collapse have occurred in patients receiving therapeutic doses of diazepam and buprenorphine [8]. Animal studies confirmed the potential dangers of the concurrent administration of buprenorphine and benzodiazepines [9,10]. The findings in the current series further emphasised the possible dangers of diverted intravenous abuse of buprenorphine. The danger was apparent even where the post-mortem blood levels were found to be within the therapeutic range [3]. It underscored the danger of concurrent administration of buprenorphine with benzodiazepines. The mechanism of interaction between buprenorphine and benzodiazepines appeared not to be dose-dependent. Current data suggested that buprenorphine could be life-threatening without overdosage when concurrently used with other psychotropic drugs [5,11]. The small sample size of the present study failed to demonstrate any cut-off for the post-mortem blood buprenorphine level for which death could be attributed to mixed drug reaction. Certification of the cause of death after autopsy therefore depended on balancing the significance of anatomical pathology and toxicological findings and becomes a matter of opinion. On the balance of probability, it was usually more defensible and sensible to attribute cause of death to evident pathology rather than evoke reasonable doubt.
There were several limitations in this study, mainly inherent in coronial casework. Firstly, coronial casework dealt with a skewed sample population, which did not necessarily reflect the general population. Secondly, there was already a selection bias in which cases were subjected to autopsies. Coronial cases that subsequently revealed significant medical history would not be autopsied unless the circumstances indicate otherwise. Requests for toxicological analyses also depended greatly on circumstances of death and autopsy findings. Similarly, toxicological screenings were also carried out in stages, based on the same variables. Thirdly, the sample size for this study was small, hence limiting the power to discern statistical significance in any observed differences. 5. Conclusion In summary, buprenorphine has been approved as substitution therapy for opioid dependence in Singapore. There was a sharp increase in the incidence of buprenorphine related deaths in the first 2 years since its availability in 2002. All fatalities in this period involved males, many who were known drug abusers. In addition, most were Chinese and under the age of 40 years. This study highlighted that concurrent administration of buprenorphine and benzodiazepines was potentially fatal. The diversion of sublingual buprenorphine preparations for intravenous use presented an additional danger. The medical community must be alerted to these potential dangers. The relevant authorities and government agencies must also act promptly to regulate the prescription of buprenorphine, in order to prevent further fatalities from the misuse of buprenorphine. Acknowledgements The authors thank the State Coroner of Singapore for permission to report these cases. The main author wishes to thank colleagues at the Centre for Forensic Medicine for permission to review their cases. References
[1] Y.J. Yao, T.L. Ng, D.S.T. Lo, P.C.T. Eng, New approach to emergency toxicology—a case study, TIAFT Bull. 30 (4) (2000) 6–7. [2] Y.J. Yao, D.S.T. Lo, J.Y. Sim, T.H. Koh, Analysis of buprenorphine and norbuprenorphine in blood and urine by liquid chromatography-electrospray-mass spectrometry (LC-ES-MS), in: Presented in the 43rd The International Association of Forensic Toxicologists Meeting, 2005. [3] J.P. de los Cobos, S. Martin, A. Etcheberrigaray, J. Trujols, F. Batlle, A. Tejero, et al., A controlled trial of daily versus thrice-weekly buprenorphine administration for the treatment of opioid dependence, Drug Alcohol Depend. 59 (3) (2000) 223–233. [4] FDA Talk Paper T02-38 October 8, 2002, http://www.fda.gov/bbs/topics/ ANSWERS/2002/ANS01165.html, accessed on 11 July 2005. [5] A. Traqui, P. Kintz, B. Ludes, Buprenorphine-related deaths among drug addicts in France: a report on 20 fatalities, J. Anal. Toxicol. 22 (6) (1998) 430–434.
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S.H. Lai et al. / Forensic Science International 162 (2006) 80–86 [9] P.N. Gueye, S.W. Borron, P. Risede, C. Monier, F. Buneaux, M. Debray, F.J. Baud, Buprenorphine and midazolam act in combination to depress respiration in rats, Toxicol. Sci. 65 (2002) 107–114. [10] S. Nielson, D.A. Taylor, The effect of buprenorphine and benzodiazepines on respiration in rats, Drug Alcohol Depend. 79 (1) (2005) 95–101. [11] P. Kintz, Deaths involving buprenorphine: a compendium of French cases, Forensic Sci. Int. 121 (2001) 65–69.
[6] J.W. Downing, N.M. Goodwin, J. Hicks, The respiratory depressive effects of intravenous buprenorphine in patients in an intensive care unit, S. Afr. Med. J. 55 (25) (1979) 1023–1027. [7] R.C. Heel, R.N. Brogden, T.M. Speight, G.S. Avery, Buprenorphine: a review of its pharmacological properties and therapeutic efficacy, Drugs 17 (2) (1979) 81–110. [8] Buprenex1, Product Info 2001.
