J Forensic Sci, November 2014, Vol. 59, No. 6 doi: 10.1111/1556-4029.12489 Available online at: onlinelibrary.wiley.com
CASE REPORT TOXICOLOGY
Iain M. McIntyre,1 Ph.D.; Phyllis Mallett,1 B.S.; Christopher G. Burton,1; and Jacquelyn Morhaime,1 M.D.
Acute Benztropine Intoxication and Fatality
ABSTRACT: A woman was found unresponsive with an empty bottle of Cogentinâ prescribed to another. Admitted to an area hospital, her
condition steadily declined until death 29 h after admission. Following toxicological screening on hospital (admission) whole blood, the only significant compound detected was benztropine. Benztropine was confirmed at 0.28 mg/L – the highest antemortem blood concentration recorded in a case of toxicity or fatality uniquely associated with benztropine. A second serum antemortem specimen showed a benztropine concentration of 0.19 mg/L. Despite over 24 h in the hospital, benztropine was also found in the postmortem specimens collected at autopsy. Peripheral blood, central blood, liver, and gastric concentrations were 0.47 mg/L, 0.36 mg/L, 9.6 mg/kg, and 44 mg, respectively. These results indicate that benztropine exhibited a potential difference between whole-blood and serum (plasma) concentrations. Additionally, in consideration of literature data, benztropine was found indicative of a compound prone to at least some postmortem redistribution.
KEYWORDS: forensic science, benztropine, fatality, antemortem and postmortem concentrations, postmortem redistribution Benztropine (Cogentinâ) is an anticholinergic drug used for over 60 years in the treatment of Parkinson’s disease, and the management of pseudo-parkinsonism and dystonia side effects often associated with neuroleptic drug treatment (1,2). Doses may be titrated in increments of 0.5 mg to a maximum dose of 6 mg, or until optimal results are obtained, at 5–6 day intervals. It is available in tablets (0.5, 1 and 2 mg) and injectable solution at 1 mg/mL. A plasma benztropine concentration of 6.7 lg/L was found at 12 h postdose following a 2 mg dose (3), and plasma concentrations were reported to range from 79.5 to 126 lg/L (average 99 lg/L) after chronic treatment with 4 mg daily (4). Adverse effects and signs of toxicity have been described to include dizziness, drowsiness, disorientation, blurred vision, dry mouth, urinary retention and constipation (2). In overdose, the symptoms are typical of those described in antihistamine or atropine overdosage and include central nervous system depression, anticholinergic manifestations, hyperpyrexia, dystonic reaction, delirium, coma and shock (5–11). There are currently no published data evaluating the relationship between whole-blood and serum (plasma) benztropine concentrations. Despite this lack of information, fatalities due to benztropine alone have been reported with postmortem blood concentrations ranging from as low as 183 lg/L (0.183 mg/L) (12–16). This is only marginally higher than chronic therapeutic plasma concentrations (4). Furthermore, there are indications of potential for benztropine postmortem redistribution (PMR), as the heart (or central) blood to peripheral blood (C/P) ratio has been shown to range from 0.6 to 7.7 (17,18).
The case reported herein examines an overdose admitted to the hospital, where both antemortem (whole-blood and serum) and postmortem benztropine concentrations were determined, and presents interpretation and discussion of the data considering potential for PMR. Case Report The decedent was a 50-year-old woman with two prior suicide attempts via medication overdose. She was found unresponsive, in her bedroom at a board and care facility where she was under care for psychiatric concerns. Next to her was an empty bottle of Cogentinâ, which had been prescribed to one of the other residents. She was transported to the hospital, where she was noted to be displaying symptoms of an anticholinergic reaction, including mydriasis, warm skin, and sinus tachycardia – initial heart rate was in the 140s. She was admitted and remained unresponsive, with a diagnosis of acute toxic encephalopathy due to medication overdose. Her condition deteriorated, and she was pronounced dead the following day – 29 h after admission. There was no medical intervention due to a “do not resuscitate” status. The only plan for her was supportive care and observation. The autopsy, apart from showing evidence of the recent medical management, was grossly unremarkable. Despite her hospital course, there were no obvious postmortem signs related to the medication overdose, not even hypoxic/ischemic changes to her brain. The autopsy was performed about 17 h after her death.
Methods 1
County of San Diego Medical Examiner’s Office, 5570 Overland Avenue, Suite 101, San Diego, CA 92123. Received 21 May 2013; and in revised form 9 Aug. 2013; accepted 24 Aug. 2013. © 2014 American Academy of Forensic Sciences
Postmortem Specimen Collection All postmortem specimens analyzed were collected at autopsy at the San Diego County Medical Examiner’s Office. Peripheral 1675
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blood was drawn from the left common iliac vein (blood returning from the leg and visually identified in the pelvis at autopsy) and stored in standard glass tubes containing sodium fluoride (100 mg) and potassium oxalate (20 mg). Central blood was collected from the intrapericardial inferior vena cava and placed in identical tubes. A section of the right lobe of liver was collected and stored in a sterile four ounce container without preservative. Total gastric contents (305 g) were also collected into a sterile container without preservative. All samples were stored at 4°C until analyzed. Toxicology A complete toxicological screening regimen was requested and performed on the antemortem (admission) whole-blood specimen. The blood was screened for alcohol and volatile compounds with headspace gas chromatography-flame ionization detection (GC-FID), drugs of abuse by enzyme-linked immunosorbent assay (ELISA: cocaine metabolite, opiates, methamphetamine, benzodiazepines, cannabinoids, fentanyl, synthetic cannabinoids, oxycodone, methadone, zolpidem, carisoprodol, and buprenorphine) (Immunalysis Inc., Pomona, CA), an alkaline drug screen by gas chromatography-mass spectrometry (GCMS) following solid-phase extraction, and an acid/neutral drug screen with high-performance liquid chromatography (HPLC) – photodiode array detection following specimen precipitation with acetonitrile. Benztropine, when detected by the alkaline drug screen, was subsequently confirmed and quantified in the antemortem whole-blood and postmortem specimens by a specific gas chromatography-nitrogen phosphorus detection (GC-NPD) procedure.
Benztropine Confirmation and Quantitation Analysis Materials Benztropine standards and controls were purchased from Alltech (State College, PA) and Ceriliant (Austin, TX) in methanol dissolved stock at a concentration of 1 mg/mL, and separate lot numbers were used for calibrators and controls. The internal standard used was cyclizine (Burroughs-Wellcome, Kirkland, QC, Canada). Cyclizine was prepared in 1.0 mg/mL stock in methanol. Working stock solutions of benztropine were prepared in deionized (DI) water at a concentration of 1.0 mg/L for both the standard and control. The cyclizine was diluted with DI water to make 5.0 mg/L working solution. 1-chlorobutane and ethyl acetate were manufactured by OmniSolv (VWR International, Radnor, PA). Concentrated hydrochloric acid was manufactured by Aristar (VWR International), and the concentrated ammonium hydroxide was from EMD (VWR International). Sodium sulfate (anhydrous, granular ACS grade) was obtained from Sigma-Aldrich Chemical (St. Louis, MO). Extraction Benztropine was first detected in an alkaline drug screen by gas chromatography-mass spectrometry (GC-MS) using a solidphase extraction of blood, method previously described (19). The drug was then quantitated on a gas chromatograph with a selective nitrogen–phosphorous detector (NPD). Five-point calibration curves were obtained by making calibrators from the working benztropine solutions in the concentrations of 0.10, 0.25, 0.50, 1.0, and 2.0 mg/L. Two controls were made from a
separate working stock with a different lot number than the calibrators in concentrations of 0.5 and 1.5 mg/L. A blood curve was used for blood analyses containing blank porcine blood as the matrix. A liver curve was constructed using porcine liver homogenate for the matrix of liver specimens. (Liver homogenates were prepared by making a 1:1 dilution with deionized water and homogenized using either a commercial blender or dispersion mixer.) Samples were all extracted using a method modified after that of Foerster et al. (20). For all samples, a minimum of two separate sample dilutions were used and placed in separate tubes. Any sample added that was less than 1 mL had its difference in volume made up with blank porcine blood. Liver specimens were homogenized by taking 15–20 g of liver specimen and blending it with an equal amount of DI water to create a 0.5 g/ mL homogenate. 1 mL of this homogenate was then pipetted into a tube and diluted to 10 mL with DI water to create a 0.05 g/mL liver homogenate. All blood specimens were run on a blood curve with its own calibrators, controls, blank and negative, and all liver samples were run with their matching matrixes as well. To each tube, 1 mL of their respective matrix (porcine blood or porcine liver) was added. Each tube was diluted to 5 mL with DI water and vortexed for 10 sec. 50 lL of cyclizine working solution was added to each tube except blanks, and the tubes were vortexed again for 10 sec. Fresh concentrated ammonium hydroxide (1 mL) was then added to each tube, and tubes were vortexed again for 10 sec. Tubes then had 6 mL of 1-chlorobutane added, were capped, and then extracted by rotation for 30 min. When finished, tubes were centrifuged at approximately 2400 9 g for 5 min. Any emulsion still present after centrifugation was eliminated by the addition of sodium sulfate in necessary quantities. All tubes were centrifuged for 5 more minutes at 2400 9 g, and then, the top organic solvent (1-chlorobutane) phase was extracted by pipette into a clean glass tube. Extracted organic layers were put into screw cap tubes, and 3.5 mL of 1N HCl was added to each tube. Tubes were capped and extracted by rotation for 30 min. Following extraction by rotation and centrifugation at 2400 9 g for 5 min, the organic 1-chlorobutane layer was aspirated to waste. A 1 mL aliquot of concentrated ammonium hydroxide was added to the remaining acid layer of each tube and vortexed for 10 sec. Then to each tube, 3 mL of 1-chlorobutane was added, tubes were capped, and extracted by rotation for 30 min. The tubes were then centrifuged for 15 min at 2400 9 g, and the top organic layer was extracted carefully to clean culture tubes. The culture tubes solvent was evaporated off at room temperature under nitrogen to dryness. Dry extracts were reconstituted with 100 lL of ethyl acetate and vortexed for 10 sec. Extracts were then transferred to autosampler vials fitted with glass volume inserts. All calibrators were within 20% of the target concentration when they were back calculated. A calibration curve was constructed from a minimum of four nonzero points. The calibration used a linear regression fit (r2 ≥ 0.99). Both positive control samples were calculated to known values of 0.5 or 1.5 mg/L. The liver homogenate specimen was diluted so the concentration would fall within the range of the calibration curve. The limit of detection was 0.05 mg/L, and the limit of quantification, determined by the lowest calibration concentration, was 0.10 mg/L. The 0.50 and 1.5 mg/L blood controls measured 0.47 0.02 mg/L (mean standard deviation) and 1.6 0.16 mg/L (mean standard deviation) over three separate analytical determinations, respectively.
MCINTYRE ET AL.
Instrumentation Benztropine specimens were analyzed using a HP 5890 Series II Plus Gas Chromatograph using a DB-1 (15 m 9 0.252 mm 9 0.25 lm) column from Agilent (Santa Clara, CA) and a nitrogen–phosphorous bead detector from Agilent. Helium was the carrier gas and had a flow rate of 1.2 mL/min. For all samples, the inlet temperature was set to 250°C and the detector set at 280°C. For blood and liver samples, 1 lL of sample was injected on the column, and after 30 sec, the GC started its oven ramp. The oven started at 50°C, and the ramp was an increase of 35°C/min for 4.5 min. After 4.5 min, the oven temperature remained constant at 275°C until the end of the run. Total runtime after injection was 13.5 min. The cyclizine internal standard was seen at about 5.8 min. Benztropine had a retention time of about 6.6 min within a window of 1% and a relative retention time (RRT) of 1.15. The analytical procedure has been employed in this laboratory for many years without any known co-eluting compounds at the retention (or RRT) time of benztropine. Results and Discussion Full toxicological screening detected only benztropine and a trace of ibuprofen (1.0 in drugs that do not redistribute. In addition, resuscitation attempts may result in a C/P ratio
CASE REPORT TOXICOLOGY
Iain M. McIntyre,1 Ph.D.; Phyllis Mallett,1 B.S.; Christopher G. Burton,1; and Jacquelyn Morhaime,1 M.D.
Acute Benztropine Intoxication and Fatality
ABSTRACT: A woman was found unresponsive with an empty bottle of Cogentinâ prescribed to another. Admitted to an area hospital, her
condition steadily declined until death 29 h after admission. Following toxicological screening on hospital (admission) whole blood, the only significant compound detected was benztropine. Benztropine was confirmed at 0.28 mg/L – the highest antemortem blood concentration recorded in a case of toxicity or fatality uniquely associated with benztropine. A second serum antemortem specimen showed a benztropine concentration of 0.19 mg/L. Despite over 24 h in the hospital, benztropine was also found in the postmortem specimens collected at autopsy. Peripheral blood, central blood, liver, and gastric concentrations were 0.47 mg/L, 0.36 mg/L, 9.6 mg/kg, and 44 mg, respectively. These results indicate that benztropine exhibited a potential difference between whole-blood and serum (plasma) concentrations. Additionally, in consideration of literature data, benztropine was found indicative of a compound prone to at least some postmortem redistribution.
KEYWORDS: forensic science, benztropine, fatality, antemortem and postmortem concentrations, postmortem redistribution Benztropine (Cogentinâ) is an anticholinergic drug used for over 60 years in the treatment of Parkinson’s disease, and the management of pseudo-parkinsonism and dystonia side effects often associated with neuroleptic drug treatment (1,2). Doses may be titrated in increments of 0.5 mg to a maximum dose of 6 mg, or until optimal results are obtained, at 5–6 day intervals. It is available in tablets (0.5, 1 and 2 mg) and injectable solution at 1 mg/mL. A plasma benztropine concentration of 6.7 lg/L was found at 12 h postdose following a 2 mg dose (3), and plasma concentrations were reported to range from 79.5 to 126 lg/L (average 99 lg/L) after chronic treatment with 4 mg daily (4). Adverse effects and signs of toxicity have been described to include dizziness, drowsiness, disorientation, blurred vision, dry mouth, urinary retention and constipation (2). In overdose, the symptoms are typical of those described in antihistamine or atropine overdosage and include central nervous system depression, anticholinergic manifestations, hyperpyrexia, dystonic reaction, delirium, coma and shock (5–11). There are currently no published data evaluating the relationship between whole-blood and serum (plasma) benztropine concentrations. Despite this lack of information, fatalities due to benztropine alone have been reported with postmortem blood concentrations ranging from as low as 183 lg/L (0.183 mg/L) (12–16). This is only marginally higher than chronic therapeutic plasma concentrations (4). Furthermore, there are indications of potential for benztropine postmortem redistribution (PMR), as the heart (or central) blood to peripheral blood (C/P) ratio has been shown to range from 0.6 to 7.7 (17,18).
The case reported herein examines an overdose admitted to the hospital, where both antemortem (whole-blood and serum) and postmortem benztropine concentrations were determined, and presents interpretation and discussion of the data considering potential for PMR. Case Report The decedent was a 50-year-old woman with two prior suicide attempts via medication overdose. She was found unresponsive, in her bedroom at a board and care facility where she was under care for psychiatric concerns. Next to her was an empty bottle of Cogentinâ, which had been prescribed to one of the other residents. She was transported to the hospital, where she was noted to be displaying symptoms of an anticholinergic reaction, including mydriasis, warm skin, and sinus tachycardia – initial heart rate was in the 140s. She was admitted and remained unresponsive, with a diagnosis of acute toxic encephalopathy due to medication overdose. Her condition deteriorated, and she was pronounced dead the following day – 29 h after admission. There was no medical intervention due to a “do not resuscitate” status. The only plan for her was supportive care and observation. The autopsy, apart from showing evidence of the recent medical management, was grossly unremarkable. Despite her hospital course, there were no obvious postmortem signs related to the medication overdose, not even hypoxic/ischemic changes to her brain. The autopsy was performed about 17 h after her death.
Methods 1
County of San Diego Medical Examiner’s Office, 5570 Overland Avenue, Suite 101, San Diego, CA 92123. Received 21 May 2013; and in revised form 9 Aug. 2013; accepted 24 Aug. 2013. © 2014 American Academy of Forensic Sciences
Postmortem Specimen Collection All postmortem specimens analyzed were collected at autopsy at the San Diego County Medical Examiner’s Office. Peripheral 1675
1676
JOURNAL OF FORENSIC SCIENCES
blood was drawn from the left common iliac vein (blood returning from the leg and visually identified in the pelvis at autopsy) and stored in standard glass tubes containing sodium fluoride (100 mg) and potassium oxalate (20 mg). Central blood was collected from the intrapericardial inferior vena cava and placed in identical tubes. A section of the right lobe of liver was collected and stored in a sterile four ounce container without preservative. Total gastric contents (305 g) were also collected into a sterile container without preservative. All samples were stored at 4°C until analyzed. Toxicology A complete toxicological screening regimen was requested and performed on the antemortem (admission) whole-blood specimen. The blood was screened for alcohol and volatile compounds with headspace gas chromatography-flame ionization detection (GC-FID), drugs of abuse by enzyme-linked immunosorbent assay (ELISA: cocaine metabolite, opiates, methamphetamine, benzodiazepines, cannabinoids, fentanyl, synthetic cannabinoids, oxycodone, methadone, zolpidem, carisoprodol, and buprenorphine) (Immunalysis Inc., Pomona, CA), an alkaline drug screen by gas chromatography-mass spectrometry (GCMS) following solid-phase extraction, and an acid/neutral drug screen with high-performance liquid chromatography (HPLC) – photodiode array detection following specimen precipitation with acetonitrile. Benztropine, when detected by the alkaline drug screen, was subsequently confirmed and quantified in the antemortem whole-blood and postmortem specimens by a specific gas chromatography-nitrogen phosphorus detection (GC-NPD) procedure.
Benztropine Confirmation and Quantitation Analysis Materials Benztropine standards and controls were purchased from Alltech (State College, PA) and Ceriliant (Austin, TX) in methanol dissolved stock at a concentration of 1 mg/mL, and separate lot numbers were used for calibrators and controls. The internal standard used was cyclizine (Burroughs-Wellcome, Kirkland, QC, Canada). Cyclizine was prepared in 1.0 mg/mL stock in methanol. Working stock solutions of benztropine were prepared in deionized (DI) water at a concentration of 1.0 mg/L for both the standard and control. The cyclizine was diluted with DI water to make 5.0 mg/L working solution. 1-chlorobutane and ethyl acetate were manufactured by OmniSolv (VWR International, Radnor, PA). Concentrated hydrochloric acid was manufactured by Aristar (VWR International), and the concentrated ammonium hydroxide was from EMD (VWR International). Sodium sulfate (anhydrous, granular ACS grade) was obtained from Sigma-Aldrich Chemical (St. Louis, MO). Extraction Benztropine was first detected in an alkaline drug screen by gas chromatography-mass spectrometry (GC-MS) using a solidphase extraction of blood, method previously described (19). The drug was then quantitated on a gas chromatograph with a selective nitrogen–phosphorous detector (NPD). Five-point calibration curves were obtained by making calibrators from the working benztropine solutions in the concentrations of 0.10, 0.25, 0.50, 1.0, and 2.0 mg/L. Two controls were made from a
separate working stock with a different lot number than the calibrators in concentrations of 0.5 and 1.5 mg/L. A blood curve was used for blood analyses containing blank porcine blood as the matrix. A liver curve was constructed using porcine liver homogenate for the matrix of liver specimens. (Liver homogenates were prepared by making a 1:1 dilution with deionized water and homogenized using either a commercial blender or dispersion mixer.) Samples were all extracted using a method modified after that of Foerster et al. (20). For all samples, a minimum of two separate sample dilutions were used and placed in separate tubes. Any sample added that was less than 1 mL had its difference in volume made up with blank porcine blood. Liver specimens were homogenized by taking 15–20 g of liver specimen and blending it with an equal amount of DI water to create a 0.5 g/ mL homogenate. 1 mL of this homogenate was then pipetted into a tube and diluted to 10 mL with DI water to create a 0.05 g/mL liver homogenate. All blood specimens were run on a blood curve with its own calibrators, controls, blank and negative, and all liver samples were run with their matching matrixes as well. To each tube, 1 mL of their respective matrix (porcine blood or porcine liver) was added. Each tube was diluted to 5 mL with DI water and vortexed for 10 sec. 50 lL of cyclizine working solution was added to each tube except blanks, and the tubes were vortexed again for 10 sec. Fresh concentrated ammonium hydroxide (1 mL) was then added to each tube, and tubes were vortexed again for 10 sec. Tubes then had 6 mL of 1-chlorobutane added, were capped, and then extracted by rotation for 30 min. When finished, tubes were centrifuged at approximately 2400 9 g for 5 min. Any emulsion still present after centrifugation was eliminated by the addition of sodium sulfate in necessary quantities. All tubes were centrifuged for 5 more minutes at 2400 9 g, and then, the top organic solvent (1-chlorobutane) phase was extracted by pipette into a clean glass tube. Extracted organic layers were put into screw cap tubes, and 3.5 mL of 1N HCl was added to each tube. Tubes were capped and extracted by rotation for 30 min. Following extraction by rotation and centrifugation at 2400 9 g for 5 min, the organic 1-chlorobutane layer was aspirated to waste. A 1 mL aliquot of concentrated ammonium hydroxide was added to the remaining acid layer of each tube and vortexed for 10 sec. Then to each tube, 3 mL of 1-chlorobutane was added, tubes were capped, and extracted by rotation for 30 min. The tubes were then centrifuged for 15 min at 2400 9 g, and the top organic layer was extracted carefully to clean culture tubes. The culture tubes solvent was evaporated off at room temperature under nitrogen to dryness. Dry extracts were reconstituted with 100 lL of ethyl acetate and vortexed for 10 sec. Extracts were then transferred to autosampler vials fitted with glass volume inserts. All calibrators were within 20% of the target concentration when they were back calculated. A calibration curve was constructed from a minimum of four nonzero points. The calibration used a linear regression fit (r2 ≥ 0.99). Both positive control samples were calculated to known values of 0.5 or 1.5 mg/L. The liver homogenate specimen was diluted so the concentration would fall within the range of the calibration curve. The limit of detection was 0.05 mg/L, and the limit of quantification, determined by the lowest calibration concentration, was 0.10 mg/L. The 0.50 and 1.5 mg/L blood controls measured 0.47 0.02 mg/L (mean standard deviation) and 1.6 0.16 mg/L (mean standard deviation) over three separate analytical determinations, respectively.
MCINTYRE ET AL.
Instrumentation Benztropine specimens were analyzed using a HP 5890 Series II Plus Gas Chromatograph using a DB-1 (15 m 9 0.252 mm 9 0.25 lm) column from Agilent (Santa Clara, CA) and a nitrogen–phosphorous bead detector from Agilent. Helium was the carrier gas and had a flow rate of 1.2 mL/min. For all samples, the inlet temperature was set to 250°C and the detector set at 280°C. For blood and liver samples, 1 lL of sample was injected on the column, and after 30 sec, the GC started its oven ramp. The oven started at 50°C, and the ramp was an increase of 35°C/min for 4.5 min. After 4.5 min, the oven temperature remained constant at 275°C until the end of the run. Total runtime after injection was 13.5 min. The cyclizine internal standard was seen at about 5.8 min. Benztropine had a retention time of about 6.6 min within a window of 1% and a relative retention time (RRT) of 1.15. The analytical procedure has been employed in this laboratory for many years without any known co-eluting compounds at the retention (or RRT) time of benztropine. Results and Discussion Full toxicological screening detected only benztropine and a trace of ibuprofen (1.0 in drugs that do not redistribute. In addition, resuscitation attempts may result in a C/P ratio
