Journal of Analytical Toxicology Advance Access published April 26, 2015 Journal of Analytical Toxicology 2015;1 – 5 doi:10.1093/jat/bkv043
Case Report
An Acute Acetyl Fentanyl Fatality: A Case Report With Postmortem Concentrations Iain M. McIntyre*, Amber Trochta, Ray D. Gary, Mark Malamatos and Jonathan R. Lucas County of San Diego Medical Examiner’s Office, 5570 Overland Ave., Suite 101, San Diego, CA 92123, USA *Author to whom correspondence should be addressed. Email: [email protected]
In this case report, we present an evaluation of the distribution of postmortem concentrations of acetyl fentanyl in a fatality attributed to the drug. A young man who had a history of heroin abuse was found deceased at his parents’ home. Toxicology testing, which initially screened positive for fentanyl by ELISA, subsequently confirmed acetyl fentanyl by gas chromatography– mass spectrometry specific ion monitoring (GC–MS SIM) analysis following liquid –liquid extraction. No other drugs or medications, including fentanyl, were detected. The acetyl fentanyl peripheral blood concentration was quantified at 260 ng/mL compared with the central blood concentration of 250 ng/mL. The liver concentration was 1,000 ng/kg, the vitreous was 240 ng/mL and the urine was 2,600 ng/mL. The cause of death was certified due to acute acetyl fentanyl intoxication, and the manner of death was certified as an accident.
Introduction Acetyl fentanyl [N-(1-phenethylpiperidin-4-yl)-N-phenylacetamide] has been encountered as an illicitly used narcotic in the USA since 2013 (1). Acetyl fentanyl and fentanyl are both synthetic opioids, with acetyl fentanyl being the N-acetyl version of fentanyl (Figure 1). As an analgesic, acetyl fentanyl—which has no recognized medical use—has been reported to be 5–15 times more potent than heroin (2), 15 times more than morphine and about 3-fold less potent than that of fentanyl (3). Although the pharmacological effects of acetyl fentanyl have not been specifically investigated clinically in humans (3), fentanyl-like substances have been generally associated with euphoria, altered mood, drowsiness, miosis, cough suppression, constipation and respiratory depression. Typically abused intravenously, acetyl fentanyl has been attributed to 14 deaths in Rhode Island (1), numerous cases in Pennsylvania (4) and 3 deaths in North Carolina (5). Other fatal and nonfatal acetyl fentanyl cases may be mistakenly attributed to heroin because patients present as if experiencing a heroin overdose, and respond similarly (although a larger dose may be required) to treatment with naloxone (6, 7). Despite an increasing frequency of reports, the determination of biological concentrations (antemortem or postmortem) is rare. There are currently no published data, although the acute overdoses from North Carolina evidently exhibited blood and urine concentrations of 330 – 410 ng/mL and 1,000–5,700 ng/mL, respectively (8). As a number of fentanyl analogs demonstrate substantial crossreactivity for the fentanyl antibody on enzyme-linked immunosorbent assay (ELISA) (9, 10), acetyl fentanyl has been detected by routine fentanyl screening tests (1). A liquid chromatography – mass spectrometry (LC – MS) confirmation procedure has been described and utilized for the analysis of urine concentrations following intravenous acetyl fentanyl administration to rats (11).
In this report, for the first time, postmortem concentrations are described for peripheral blood, central blood, liver, vitreous humor and urine in a death certified solely to acetyl fentanyl. An analytical procedure was developed by minor modification of a gas chromatography –mass spectrometry (GC – MS) specific ion monitoring (SIM) method for fentanyl analysis (12). Methods Case report A 24-year-old man (height 67 inches and weight 152 pounds) lived at and was the assistant manager of a sober living residence in San Diego. He was visiting his parents’ home for a weekend visit. His parents last saw him at 2030 h on 29 November 2014 when he left to go visit friends. He returned home at an unknown time during the night. At approximately 0850 hours on the morning of 30 November 2014, his mother found him unresponsive behind the closed bedroom closet door with an uncapped syringe with a bent needle, syringe cap and rubber tourniquet. Emergency 9-1-1 was called, and he was declared dead at the scene. The syringe was collected as evidence. He had a history of ‘potential’ heroin abuse. He had previously overdosed twice in the closet at home with what was believed to be heroin. A complete autopsy was conducted on 1 December 2014 at 0945 h, approximately 25 h after he was found, and documented three apparent recent punctures in the left forearm and antecubital fossa. The lungs were edematous and congested (right 610 g and left 580 g). Microscopically, the lungs and a left arm vein contained small amounts of foreign material consistent with chronic/prior intravenous drug abuse. There were no other significant findings. Postmortem specimen collection All specimens analyzed were collected at autopsy at the San Diego County Medical Examiner’s Office. Peripheral blood (20 mL) 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 directly from the heart and placed in identical tubes. Sections of the right lobe of liver were collected and stored in an opaque plastic four ounce container without preservative. Vitreous humor samples were withdrawn from the eyes with a syringe and stored in a glass tube without preservative. Urine was collected into in an opaque plastic four ounce container without preservative. All samples were stored at 48C until analyzed within 6 weeks of collection.
Toxicology A comprehensive toxicological screening regimen was performed. Postmortem blood was screened for alcohol and volatile
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Chemical structures.
compounds (GC-FID headspace), 12 drugs of abuse panel by ELISA (cocaine metabolite, opiates, methamphetamine, benzodiazepines, cannabinoids, fentanyl, phencyclidine, oxycodone, methadone, zolpidem, carisoprodol and buprenorphine; Immunalysis, Inc., Pomona, CA, USA), an alkaline drug screen by GC –MS following solid-phase extraction and an acid/neutral drug screen with HPLC-photodiode array detection following specimen precipitation with acetonitrile. An additional screen (GC– MS) was also performed on the syringe exhibit that was collected from the scene. Following routine practice, the positive result was confirmed and quantified by a subsequent and specific technique.
Acetyl fentanyl confirmation analysis Materials All solvents and chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA) and were analytical grade or better. Test tubes made of borosilicate glass used for all phases of the extraction procedure were purchased through VWR International (Radnor, PA, USA). The acetyl fentanyl drug standard used in the calibration formulations and the D5-fentanyl internal standard were both purchased from Cerilliant Corporation (Round Rock, TX, USA). Extraction Acetyl fentanyl was confirmed and quantified utilizing minor modifications to a previously described procedure for fentanyl using gas chromatography (GC) coupled with a mass spectrometer (MS) SIM procedure (12). The analysis included whole-blood ( porcine) calibrators (100, 200, 500, 750 and 1,000 ng/mL), case samples (whole-blood, liver, vitreous and urine), positive controls and negative controls that were subjected to an alkaline liquid/liquid extraction procedure. Case specimens were extracted using appropriate dilutions (as required) to ensure that the quantitation was within the range of the calibration curve. To 1 mL of specimen, 1 mL of deionized water was added and vortexed. Next, 50 mL of working internal standard (D5-fentanyl, 10 mg/mL) was added and vortexed. Samples were then made alkaline by the addition of 1 mL of concentrated ammonium hydroxide before being vortexed again. About 6 mL of 1-chlorobutane was then added before tubes were capped and mixed on a mechanical 2 McIntyre et al.
rocker for 10 min. Samples were then centrifuged for 5 min at 2,400 g. Approximately 200 mg of sodium sulfate was added to each tube to suppress emulsions and the tubes were centrifuged for another 5 minutes at 2,400 g. The top organic layer was then transferred to new test tubes. Two mL of 1.0 N hydrochloric acid was added and tubes were mixed for 10 min. The tubes were then centrifuged for 5 min at 2,400 g before the top organic layer was aspirated to waste. The remaining aqueous portion was made alkaline with 1 mL of concentrated ammonium hydroxide and vortexed. About 3 mL of 1-chlorobutane was added and tubes were mixed for 10 min. Samples were then centrifuged for 5 minutes at 2,400 g before the top organic layer was transferred to a new test tube. The organic layer was then dried under a stream of nitrogen at 378C. Samples were then reconstituted with 50 mL of ethyl acetate before being transferred to autosampler vials for analysis by GC–MS. Chromatographic conditions The following GC–MS conditions were used in the analysis. The samples (1 mL) were injected splitless into a GC – MS (7890A/ 5975C; Agilent Technologies, Santa Clara, CA, USA) equipped with a capillary column (Zebron ZB-5MS, 15 m, 0.25 mm i.d., 0.25 mm; Phenomenex, Torrance, CA, USA), and run in selective ion mode. Ions monitored for acetyl fentanyl were 231 (quantitative), 146 and 188. Internal standard ions monitored for D5-fentanyl were 250, 151 and 194. The injector temperature was 2508C and the initial oven temperature was 1008C. The oven was ramped at 208C/min to 2908C and held for 2 min. Helium was used as the carrier gas at a constant rate of 1.1 mL/min. Retention time for acetyl fentanyl was 8.78 min. Validation The limit of detection (LOD) (with the calibration curve employed) was 50 ng/mL and limit of quantitation (LOQ), determined from the lowest calibration concentration, was 100 ng/ mL. Both LOD and LOQ concentrations could be substantially lowered by the utilization of a more sensitive calibration; quantitative and qualifier ions were distinctly measurable at concentrations as low as 1.0 ng/mL. Control samples, prepared from the same source as the calibrators (but prepared independently) at 200 and 800 ng/mL in whole blood, measured 200 + 5.5 ng/ mL (mean + standard deviation; N ¼ 4) and 800 + 10 ng/mL (mean + standard deviation; N ¼ 4), respectively. Matrix effects were evaluated by extraction and analyses of comparable control specimens (200 and 800 ng/mL) prepared in water and a liver homogenate. No matrix effect was observed. Levels of 200 + 27 ng/mL (mean + standard deviation; N ¼ 4) and 780 + 26 ng/mL (mean + standard deviation; N ¼ 4) were determined for water, and 220 + 4.5 ng/mL (mean + standard deviation; N ¼ 4) and 820 + 4.5 ng/mL (mean + standard deviation; N ¼ 4) were attained for the liver homogenates. Additionally, both extracted blank (extract containing no additives) and negative control (extract containing only internal standard) specimens confirmed a lack of interference and/or contamination.
Results and discussion As described by previous investigations (1, 9, 10), acetyl fentanyl was initially detected by ELISA screening for fentanyl. The
Figure 2. Case chromatogram from GC– MS alkaline screen.
screen, established in this laboratory with 1.0 ng/mL of fentanyl as a reference, provided a positive result with 50% binding compared with a negative sample (100% binding). In the case presented, the central blood demonstrated 8% binding—a clearly positive finding. Fentanyl, however, was not detected by a confirmation method. The fentanyl GC –MS SIM method had an LOD and LOQ of 0.5 and 1.0 ng/mL, respectively (12). Furthermore, no other drugs or medications were detected by the comprehensive toxicology screening employed. Acetyl fentanyl was subsequently presumptively identified in the peripheral blood from the SWGDRUG Mass Spectral Library (http://www.swgdrug.org) following solid-phase extraction using a GC –MS alkaline screening method (13). It was confirmed with extraction, and a full mass spectral scan of a pure stock of the compound, at 18.2 min (RRT ¼ 2.19; compared with internal standard cyclizine) with significant ions of 231, 146, 188, 105, 91 and 43 (Figures 2 and 3). The acetyl fentanyl concentrations were then quantified by a specific GC – MS SIM (described above). The peripheral blood concentration was measured at 260 ng/mL compared with the
central blood concentration of 250 ng/mL. The liver concentration was 1,000 ng/kg, the vitreous was 240 ng/mL and the urine was 2,600 ng/mL. The syringe located at the scene, and collected as evidence, was also confirmed to contain only acetyl fentanyl. The central blood/peripheral blood (C/P) ratio was 0.96, and the liver/peripheral blood (L/P) ratio was 3.8 L/kg. Based on established C/P drug ratio data (14) and given recent information documenting the L/P ratio as a marker for postmortem redistribution (PMR), these data suggest that minimal potential for acetyl fentanyl PMR—L/P ratios less than 5 L/kg indicate little to no propensity toward PMR, while ratios exceeding 20 – 30 L/kg are indicative of a propensity for significant PMR (15, 16). As this deduction results from a single observation it should be viewed with caution, although these ratios are comparable to those previously reported for fentanyl (17). Unlike earlier reports describing acetyl fentanyl fatalities, no other drugs or medications were detected in this case. Various concomitant mixtures of drugs, including cocaine, other opioids, ethanol and benzodiazepines, have been identified with acetyl fentanyl in other overdoses (1). Nevertheless acetyl fentanyl, An Acute Acetyl Fentanyl Fatality 3
Figure 3. (A) Full scan mass spectrum for acetyl fentanyl from case blood extraction. (B) Comparison full scan mass spectrum for acetyl fentanyl from CaymanSpectraLibrary.
being a highly potent CNS-depressant (m-opioid receptor agonist), is liable to cause death through life-threatening hypoventilation and/or fatal respiratory depression following abuse— particularly when injected intravenously (3, 6). Consequently, the cause of death was certified due to acute acetyl fentanyl intoxication. The manner of death was certified as an accident.
Acknowledgments The authors thank the San Diego County Chief Medical Examiner, Dr Glenn Wagner, for making available case details described in this report.
References 1. MMWR. (2013) Notes from the field: acetyl fentanyl overdose fatalities— Rhode Island, March-May 2013. Morbidity and Mortality Weekly Report, 62, 703–704. 2. Higashikawa, Y., Suzuki, S. (2008) Studies on 1-(2-phenthyl)-4(N-propionylanilino)piperidine (fentanyl) and its related compounds: structure-analgesic activity relationship for fentanyl, methyl-substituted fentanyls and other analogues. Forensic Toxicology, 26, 1–5.
4 McIntyre et al.
3. Drug Enforcement Administration. (2013) Acetyl fentanyl (N-(1phenethylpiperidine-4-yl)-N-phenylamide). http://www. deadiversion.usdoj.gov/drug_chem_info/acetyl fentanyl.pdf. 4. Pennsylvania Department of Drug and Alcohol Programs. (2013) Department of Drug and Alcohol Programs Warns about Acetyl Fentanyl: Drug Caused at least 50 Fatalities This Year in Pennsylvania. Harrisburg, PA. http://www.pa.gov/ (then search: acetyl fentanyl). 5. North Carolina Department of Health and Human Services. (2014) DHHS Issues Health Advisory For Deadly New Synthetic Drug: Acetyl Fentanyl Detected In Specimens Associated With Three NC Deaths This Year. http://www.ncdhhs.gov/pressrel/2014/2014-0219_health_advisory.htm. 6. Stogner, J.M. (2014) The potential threat of acetyl fentanyl: legal issues, contaminated heroin, and acetyl fentanyl ‘disguised’ as other opioids. Annals of Emergency Medicine, 64, 637– 639. 7. Schumann, H., Erickson, T., Thompson, T.M., Zautcke, J.L., Denton, J.S. (2008) Fentanyl epidemic in Chicago, Illinois and surrounding Cook County. Clinical Toxicology, 46, 501–506. 8. Baselt, R. Personal communication. In Baselt, R.C. (ed). Preparation: Disposition of Toxic Drugs and Chemicals in Man, 11th edition. Biomedical Publications, Seal Beach, California, USA 9. Ohta, H., Suzuki, S., Ogasawara, K. (1999) Studies on fentanyl and related compounds IV. Chromatographic and spectrometric discrimination of fentanyl and its derivatives. Journal of Analytical Toxicology, 23, 280–285.
10. Wang, B.T., Colby, J.M., Wu, A.H., Lynch, K.L. (2014) Cross-reactivity of acetyl fentanyl and risperidone with a fentanyl immunoassay. Journal of Analytical Toxicology, 38, 672– 675. 11. Patton, A.L., Seely, K.A., Pulla, S., Rusch, N.J., Moran, C.L., Fantegrossi, W.E. et al. (2014) Quantitative measurement of acetyl fentanyl and acetyl norfentanyl in human urine by LC-MS/MS. Analytical Chemistry, 86, 1760–1766. 12. McIntyre, I.M., Gary, R.D., Estrada, J., Nelson, C.L. (2014) Antemortem and postmortem fentanyl concentrations: a case report. International Journal of Legal Medicine, 128, 65 –67. 13. McIntyre, I.M., Gary, R.G., Trochta, A., Stolberg, S., Stabley, R. (2014) Acute 5-(2-aminopropyl)benzofuran (5-APB) intoxication and fatality— a case report with postmortem concentrations. Journal of Analytical Toxicology; doi:10.1093/jat/bku131.
14. Dalpe-Scott, M., Degouffe, M., Garbutt, D., Drost, M. (1995) A comparison of drug concentrations in postmortem cardiac and peripheral blood in 320 cases. Canadian Society of Forensic Science Journal, 28, 113– 121. 15. McIntyre, I.M., Sherrard, J., Lucas, J. (2012) Postmortem carisoprodol and meprobamate concentrations in blood and liver: lack of significant redistribution. Journal of Analytical Toxicology, 36, 177 – 181. 16. McIntyre, I.M. (2014) Liver and peripheral blood concentration ratio (L/P) as a marker of postmortem drug redistribution: a literature review. Forensic Science, Medicine and Pathology, 10, 91 –96. 17. McIntyre, I.M., Anderson, D.T. (2012) Postmortem fentanyl concentrations: a review. Journal of Forensic Research, 3, 157. doi:10.4172/2157-7145.1000157.
An Acute Acetyl Fentanyl Fatality 5
Case Report
An Acute Acetyl Fentanyl Fatality: A Case Report With Postmortem Concentrations Iain M. McIntyre*, Amber Trochta, Ray D. Gary, Mark Malamatos and Jonathan R. Lucas County of San Diego Medical Examiner’s Office, 5570 Overland Ave., Suite 101, San Diego, CA 92123, USA *Author to whom correspondence should be addressed. Email: [email protected]
In this case report, we present an evaluation of the distribution of postmortem concentrations of acetyl fentanyl in a fatality attributed to the drug. A young man who had a history of heroin abuse was found deceased at his parents’ home. Toxicology testing, which initially screened positive for fentanyl by ELISA, subsequently confirmed acetyl fentanyl by gas chromatography– mass spectrometry specific ion monitoring (GC–MS SIM) analysis following liquid –liquid extraction. No other drugs or medications, including fentanyl, were detected. The acetyl fentanyl peripheral blood concentration was quantified at 260 ng/mL compared with the central blood concentration of 250 ng/mL. The liver concentration was 1,000 ng/kg, the vitreous was 240 ng/mL and the urine was 2,600 ng/mL. The cause of death was certified due to acute acetyl fentanyl intoxication, and the manner of death was certified as an accident.
Introduction Acetyl fentanyl [N-(1-phenethylpiperidin-4-yl)-N-phenylacetamide] has been encountered as an illicitly used narcotic in the USA since 2013 (1). Acetyl fentanyl and fentanyl are both synthetic opioids, with acetyl fentanyl being the N-acetyl version of fentanyl (Figure 1). As an analgesic, acetyl fentanyl—which has no recognized medical use—has been reported to be 5–15 times more potent than heroin (2), 15 times more than morphine and about 3-fold less potent than that of fentanyl (3). Although the pharmacological effects of acetyl fentanyl have not been specifically investigated clinically in humans (3), fentanyl-like substances have been generally associated with euphoria, altered mood, drowsiness, miosis, cough suppression, constipation and respiratory depression. Typically abused intravenously, acetyl fentanyl has been attributed to 14 deaths in Rhode Island (1), numerous cases in Pennsylvania (4) and 3 deaths in North Carolina (5). Other fatal and nonfatal acetyl fentanyl cases may be mistakenly attributed to heroin because patients present as if experiencing a heroin overdose, and respond similarly (although a larger dose may be required) to treatment with naloxone (6, 7). Despite an increasing frequency of reports, the determination of biological concentrations (antemortem or postmortem) is rare. There are currently no published data, although the acute overdoses from North Carolina evidently exhibited blood and urine concentrations of 330 – 410 ng/mL and 1,000–5,700 ng/mL, respectively (8). As a number of fentanyl analogs demonstrate substantial crossreactivity for the fentanyl antibody on enzyme-linked immunosorbent assay (ELISA) (9, 10), acetyl fentanyl has been detected by routine fentanyl screening tests (1). A liquid chromatography – mass spectrometry (LC – MS) confirmation procedure has been described and utilized for the analysis of urine concentrations following intravenous acetyl fentanyl administration to rats (11).
In this report, for the first time, postmortem concentrations are described for peripheral blood, central blood, liver, vitreous humor and urine in a death certified solely to acetyl fentanyl. An analytical procedure was developed by minor modification of a gas chromatography –mass spectrometry (GC – MS) specific ion monitoring (SIM) method for fentanyl analysis (12). Methods Case report A 24-year-old man (height 67 inches and weight 152 pounds) lived at and was the assistant manager of a sober living residence in San Diego. He was visiting his parents’ home for a weekend visit. His parents last saw him at 2030 h on 29 November 2014 when he left to go visit friends. He returned home at an unknown time during the night. At approximately 0850 hours on the morning of 30 November 2014, his mother found him unresponsive behind the closed bedroom closet door with an uncapped syringe with a bent needle, syringe cap and rubber tourniquet. Emergency 9-1-1 was called, and he was declared dead at the scene. The syringe was collected as evidence. He had a history of ‘potential’ heroin abuse. He had previously overdosed twice in the closet at home with what was believed to be heroin. A complete autopsy was conducted on 1 December 2014 at 0945 h, approximately 25 h after he was found, and documented three apparent recent punctures in the left forearm and antecubital fossa. The lungs were edematous and congested (right 610 g and left 580 g). Microscopically, the lungs and a left arm vein contained small amounts of foreign material consistent with chronic/prior intravenous drug abuse. There were no other significant findings. Postmortem specimen collection All specimens analyzed were collected at autopsy at the San Diego County Medical Examiner’s Office. Peripheral blood (20 mL) 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 directly from the heart and placed in identical tubes. Sections of the right lobe of liver were collected and stored in an opaque plastic four ounce container without preservative. Vitreous humor samples were withdrawn from the eyes with a syringe and stored in a glass tube without preservative. Urine was collected into in an opaque plastic four ounce container without preservative. All samples were stored at 48C until analyzed within 6 weeks of collection.
Toxicology A comprehensive toxicological screening regimen was performed. Postmortem blood was screened for alcohol and volatile
# The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]
Figure 1. Chemical structures.
compounds (GC-FID headspace), 12 drugs of abuse panel by ELISA (cocaine metabolite, opiates, methamphetamine, benzodiazepines, cannabinoids, fentanyl, phencyclidine, oxycodone, methadone, zolpidem, carisoprodol and buprenorphine; Immunalysis, Inc., Pomona, CA, USA), an alkaline drug screen by GC –MS following solid-phase extraction and an acid/neutral drug screen with HPLC-photodiode array detection following specimen precipitation with acetonitrile. An additional screen (GC– MS) was also performed on the syringe exhibit that was collected from the scene. Following routine practice, the positive result was confirmed and quantified by a subsequent and specific technique.
Acetyl fentanyl confirmation analysis Materials All solvents and chemicals were purchased from Fisher Scientific (Pittsburgh, PA, USA) and were analytical grade or better. Test tubes made of borosilicate glass used for all phases of the extraction procedure were purchased through VWR International (Radnor, PA, USA). The acetyl fentanyl drug standard used in the calibration formulations and the D5-fentanyl internal standard were both purchased from Cerilliant Corporation (Round Rock, TX, USA). Extraction Acetyl fentanyl was confirmed and quantified utilizing minor modifications to a previously described procedure for fentanyl using gas chromatography (GC) coupled with a mass spectrometer (MS) SIM procedure (12). The analysis included whole-blood ( porcine) calibrators (100, 200, 500, 750 and 1,000 ng/mL), case samples (whole-blood, liver, vitreous and urine), positive controls and negative controls that were subjected to an alkaline liquid/liquid extraction procedure. Case specimens were extracted using appropriate dilutions (as required) to ensure that the quantitation was within the range of the calibration curve. To 1 mL of specimen, 1 mL of deionized water was added and vortexed. Next, 50 mL of working internal standard (D5-fentanyl, 10 mg/mL) was added and vortexed. Samples were then made alkaline by the addition of 1 mL of concentrated ammonium hydroxide before being vortexed again. About 6 mL of 1-chlorobutane was then added before tubes were capped and mixed on a mechanical 2 McIntyre et al.
rocker for 10 min. Samples were then centrifuged for 5 min at 2,400 g. Approximately 200 mg of sodium sulfate was added to each tube to suppress emulsions and the tubes were centrifuged for another 5 minutes at 2,400 g. The top organic layer was then transferred to new test tubes. Two mL of 1.0 N hydrochloric acid was added and tubes were mixed for 10 min. The tubes were then centrifuged for 5 min at 2,400 g before the top organic layer was aspirated to waste. The remaining aqueous portion was made alkaline with 1 mL of concentrated ammonium hydroxide and vortexed. About 3 mL of 1-chlorobutane was added and tubes were mixed for 10 min. Samples were then centrifuged for 5 minutes at 2,400 g before the top organic layer was transferred to a new test tube. The organic layer was then dried under a stream of nitrogen at 378C. Samples were then reconstituted with 50 mL of ethyl acetate before being transferred to autosampler vials for analysis by GC–MS. Chromatographic conditions The following GC–MS conditions were used in the analysis. The samples (1 mL) were injected splitless into a GC – MS (7890A/ 5975C; Agilent Technologies, Santa Clara, CA, USA) equipped with a capillary column (Zebron ZB-5MS, 15 m, 0.25 mm i.d., 0.25 mm; Phenomenex, Torrance, CA, USA), and run in selective ion mode. Ions monitored for acetyl fentanyl were 231 (quantitative), 146 and 188. Internal standard ions monitored for D5-fentanyl were 250, 151 and 194. The injector temperature was 2508C and the initial oven temperature was 1008C. The oven was ramped at 208C/min to 2908C and held for 2 min. Helium was used as the carrier gas at a constant rate of 1.1 mL/min. Retention time for acetyl fentanyl was 8.78 min. Validation The limit of detection (LOD) (with the calibration curve employed) was 50 ng/mL and limit of quantitation (LOQ), determined from the lowest calibration concentration, was 100 ng/ mL. Both LOD and LOQ concentrations could be substantially lowered by the utilization of a more sensitive calibration; quantitative and qualifier ions were distinctly measurable at concentrations as low as 1.0 ng/mL. Control samples, prepared from the same source as the calibrators (but prepared independently) at 200 and 800 ng/mL in whole blood, measured 200 + 5.5 ng/ mL (mean + standard deviation; N ¼ 4) and 800 + 10 ng/mL (mean + standard deviation; N ¼ 4), respectively. Matrix effects were evaluated by extraction and analyses of comparable control specimens (200 and 800 ng/mL) prepared in water and a liver homogenate. No matrix effect was observed. Levels of 200 + 27 ng/mL (mean + standard deviation; N ¼ 4) and 780 + 26 ng/mL (mean + standard deviation; N ¼ 4) were determined for water, and 220 + 4.5 ng/mL (mean + standard deviation; N ¼ 4) and 820 + 4.5 ng/mL (mean + standard deviation; N ¼ 4) were attained for the liver homogenates. Additionally, both extracted blank (extract containing no additives) and negative control (extract containing only internal standard) specimens confirmed a lack of interference and/or contamination.
Results and discussion As described by previous investigations (1, 9, 10), acetyl fentanyl was initially detected by ELISA screening for fentanyl. The
Figure 2. Case chromatogram from GC– MS alkaline screen.
screen, established in this laboratory with 1.0 ng/mL of fentanyl as a reference, provided a positive result with 50% binding compared with a negative sample (100% binding). In the case presented, the central blood demonstrated 8% binding—a clearly positive finding. Fentanyl, however, was not detected by a confirmation method. The fentanyl GC –MS SIM method had an LOD and LOQ of 0.5 and 1.0 ng/mL, respectively (12). Furthermore, no other drugs or medications were detected by the comprehensive toxicology screening employed. Acetyl fentanyl was subsequently presumptively identified in the peripheral blood from the SWGDRUG Mass Spectral Library (http://www.swgdrug.org) following solid-phase extraction using a GC –MS alkaline screening method (13). It was confirmed with extraction, and a full mass spectral scan of a pure stock of the compound, at 18.2 min (RRT ¼ 2.19; compared with internal standard cyclizine) with significant ions of 231, 146, 188, 105, 91 and 43 (Figures 2 and 3). The acetyl fentanyl concentrations were then quantified by a specific GC – MS SIM (described above). The peripheral blood concentration was measured at 260 ng/mL compared with the
central blood concentration of 250 ng/mL. The liver concentration was 1,000 ng/kg, the vitreous was 240 ng/mL and the urine was 2,600 ng/mL. The syringe located at the scene, and collected as evidence, was also confirmed to contain only acetyl fentanyl. The central blood/peripheral blood (C/P) ratio was 0.96, and the liver/peripheral blood (L/P) ratio was 3.8 L/kg. Based on established C/P drug ratio data (14) and given recent information documenting the L/P ratio as a marker for postmortem redistribution (PMR), these data suggest that minimal potential for acetyl fentanyl PMR—L/P ratios less than 5 L/kg indicate little to no propensity toward PMR, while ratios exceeding 20 – 30 L/kg are indicative of a propensity for significant PMR (15, 16). As this deduction results from a single observation it should be viewed with caution, although these ratios are comparable to those previously reported for fentanyl (17). Unlike earlier reports describing acetyl fentanyl fatalities, no other drugs or medications were detected in this case. Various concomitant mixtures of drugs, including cocaine, other opioids, ethanol and benzodiazepines, have been identified with acetyl fentanyl in other overdoses (1). Nevertheless acetyl fentanyl, An Acute Acetyl Fentanyl Fatality 3
Figure 3. (A) Full scan mass spectrum for acetyl fentanyl from case blood extraction. (B) Comparison full scan mass spectrum for acetyl fentanyl from CaymanSpectraLibrary.
being a highly potent CNS-depressant (m-opioid receptor agonist), is liable to cause death through life-threatening hypoventilation and/or fatal respiratory depression following abuse— particularly when injected intravenously (3, 6). Consequently, the cause of death was certified due to acute acetyl fentanyl intoxication. The manner of death was certified as an accident.
Acknowledgments The authors thank the San Diego County Chief Medical Examiner, Dr Glenn Wagner, for making available case details described in this report.
References 1. MMWR. (2013) Notes from the field: acetyl fentanyl overdose fatalities— Rhode Island, March-May 2013. Morbidity and Mortality Weekly Report, 62, 703–704. 2. Higashikawa, Y., Suzuki, S. (2008) Studies on 1-(2-phenthyl)-4(N-propionylanilino)piperidine (fentanyl) and its related compounds: structure-analgesic activity relationship for fentanyl, methyl-substituted fentanyls and other analogues. Forensic Toxicology, 26, 1–5.
4 McIntyre et al.
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