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Journal of Analytical Toxicology, Vol. 26, January/February 2002 [CaseReport Distribution of Triazolam and (z-Hydroxytriazolam in a Fatal Intoxication Case Barry tevine 1,~,*, Alison Grieshaber 2, Joseph Pestaner 1, Karla A. Moore 1, and John E. Smialek 1 70ffice of the Chief Medical Examiner, State of Maryland, 111 Penn Street, Baltimore, Maryland 21201 and 2Division of Forensic Toxicology, Armed Forces Institute of Pathology, Washington, D.C, 20306-6000 [Abstract The case of a 77-year-old woman who was found dead in her bathtub with her head clearly above the water line is presented. The decedent had a medical history of depression, liver disease,spinal stenosis,and diabetes mellitus. An empty medication bottle of triazolam was found in the trashcan. At autopsy, no injury or evidence of drowning was found. Toxicological analysis identified triazolam at a concentration of 0.12 mg/L in the heart blood. Triazolam and cc-hydroxytriazolam were quantitated in the specimens received. The medical examiner ruled that the cause of death was triazolam intoxication and the manner of death was suicide. Introduction Triazolam is a short-acting triazolobenzodiazepine used to treat insomnia. It is sold under the trade name Halcion | and is available in 0.125-mg or 0.25-rag tablets (1). A number of pharmacokinetic studies indicate peak blood concentrations up to 0.017 mg/L following doses up to 1 mg (2). Triazolam is extensively metabolized in humans by hydroxylation and conjugation. Its major metabolite, 1-hydroxymethyltriazolam (o~-OHtriazolam), is pharmacologically active. The major urinary metabolites are glucuronide conjugates of the hydroxylated metabolites; only trace amounts of parent drug are detected in the urine (3). A number of fatalities due to triazolam with and without alcohol and other drugs have been reported (4--8). The following is a report of triazolam intoxication in which triazolam and r were quantitated in the biological specimens. Case History A 77-year-old woman was found dead in her bathtub. She was wearing a bathing suit, and her head was clearly above the water line. Police were called to the house by a neighbor, who * Disclaimer: The opinions or assertionscontained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of Defense or of the Army, Navy, or Air Force. Acknowledgment: This work was supported in part by the American Registryof Pathology. * Author to whom correspondence should be addressed. 52 also unlocked the door to allow the police to enter. The house was secure, and the decedent was in a second floor bathtub. There was an empty medication bottle of triazolam found in the trashcan with its cap on the sink counter; a note demonstrating increased somnolence was also at the scene. The decedent's medical history included depression, liver disease, spinal stenosis, and diabetes mellitus. A complete autopsy was performed, and no injury was noted. In addition, no water was identified in the sinuses, airway, or stomach, and no temporal bone hemorrhage was noted. Other than emphysema, there were no significant pulmonary findings. Moderate atherosclerotic cardiovascular disease and hepatic cirrhosis were found. The spleen was enlarged, and the sclerae were not icteric. No medication was seen in the gastric contents, which consisted of 160 mL of fluid and digested material. Based on the investigation and autopsy findings, the cause of death was pended for toxicological analysis. Experimental Triazolam and oc-OH-triazolam analysis Specimen preparation. Tissue specimens were prepared according to a previously presented method (9). TWo grams of tissue was mixed with 0.3 mL 1.0N acetic acid and centrifuged. The supernatant was removed, and the pH was adjusted to 6 by adding base. Two internal standards were used: 0.2 mg alprazolam for triazolam quantitation and 0.1 mg oc-hydroxyalprazolam-d5 for (z-OH-triazolam analysis. Internal standards were added directly to the blank, calibrators, control, and fluids prior to buffering; internal standards were added prior to the preparation of tissue homogenates and buffering. Five calibrators in drug-free blood were prepared at the following concentrations: 0.02, 0.05, 0.1, 0.2, and 0.5 rng/L. In addition, a separate 0.1-mg/L blood control was prepared. The 0.02-mg/L calibrator served as the lower limit of quantitation; the 0.5-mg/L calibrator served as the upper limit of quantitation. One milliliter of 100raM pH 6 phosphate buffer was added to 2 mL blank, calibrator, control, fluid, or tissue supernatant. After mixing and centrifuging, each solution was added to a Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission. Journal of Analytical Toxicology, Vol. 26, January/February 2002 10-mL Clean Screen| extraction column previously conditioned by sequential additions of 3 mL methanol, 3 mL deionized water, and 2 mL 100raM pH 6 phosphate buffer. The columns were washed with 3 mL deionizedwater and 2 mL 20% acetonitrile in 100mM pH 6 phosphate buffer. The columns were then dried for 5 rain and rinsed with 1 mL hexane. The compounds were eluted with 2 mL 2% ammonium hydroxide in ethyl acetate. The eluants were evaporated to dryness. Enzymatic hydrolysis. Internal standard and 1 mL of 100mM pH 6 phosphate buffer were added to urine samples. Hydrolysis was perfomedby adding ~-glucuronidase(Helixpomatia; Sigma, type H-2 crude solution) at a concentration equivalent to 2500-5000 U/mL of urine used. The mixture was vortex mixed. After ensuring that the pH was between 5.5 and 6.5, the tubes were placed in a 56~ water bath for 1.5 h. After centrifugation, the supernatant was removed and extracted as described. Derivatization. The residues were reconstituted in 50 IJL BSTFA and heated at 90~ for 15 min to produce the trimethylsilyl derivatives of the hydroxylated compounds. After cooling, 2 tJL was injected into the gas chromatograph-mass spectrometer (GC-MS). Instrumentation. A Hewlett-Packard 6890 GC attached to a 5973 mass selectivedetector.The followingconditions were used: GC column, DB-1 15-m x 0.25-ram column, 0.25-1Jmthickness; injector, splitless; injection port temperature, 255~ transfer line temperature, 255~ oven parameters, initial temperature 140~ rate #1, 25.0~ to 225~ rate #2, 12.0~ to 245~ rate #3, 30.0~ to 300~ run time, 8.9 min; carrier gas, helium; and constant pressure, 10.0 psi. The mass spectrometer was operated in the electron ionization mode. The following ions were monitored: m/z 279 for alprazolam; m/z 386 for ~-hydroxyalprazolam-d~-TMS;m/z 313, 342, and 238 for triazolam; and m/z 415, 417, and 430 for a-hydroxytriazolam-TMS. The quantitating ions for triazolam and ~-hydroxytriazolam-TMSwere m/z 313 and 415, respectively. Results A urine specimen from the presented case was tested for volatile substances, therapeutic, and abused drugs. Volatile Table I. Triazolam (T) and c~-Hydroxytriazolam (OH-T) Concentrations in the Presented Case Specimen Blood, heart (rag/L) Blood, peripheral (mg/L) Kidney (mg/kg) Liver (mg/kg) Cerebrospinal fluid (mg/L) Urine, unhydrolyzed (mg/L) Urine, hydrolyzed (mg/L) * NF = nonefound +QNS = quantity not sufficient T OH-T 0.12 0.091 0.071 0.092 0.023 0.024 QNS ~ NF* NF 0.22 0.043 NF 0.030 1.37 testing for methanol, ethanol, acetone, and isopropanol was accomplished using headspace GC. Acidic/neutral and alkaline drug testing were accomplished by GC-nitrogen-phosphorus detection. The urine was tested for morphine by radioimmunoassay and for acetaminophen, ethchlorvynol, and salicylate by color tests. No other volatile substances or therapeutic or abused drugs were detected using these techniques. Because of the suspicions of a triazolam intoxication, the urine was screened for benzodiazepines by fluorescence polarization immunoassay at a 0.1-mg/L nordiazepam cutoff. This screening test was positive. As a result, the specimens were tested for triazolam and ~-OH-triazolam. Table I lists the quantitative results in the presented case. Discussion The heart blood concentration of 0.12 mg/L in the presented case, is consistent with blood concentrations previously reported in triazolam fatalities. Bal et al. (4) reported three cases of drug intoxication involving triazolam. Blood concentrations ranged from 0.016 to 0.026 mg/L; other drugs were present in each case. Steentoft and Worm (5) measured triazolam in blood in five cases of triazolam intoxication. In one case with no ethanol present, the blood concentration was 0.11 mg/L. In four cases with ethanol greater than 0.10 g/dL present, the blood triazolam concentrations ranged from 0.04 to 0.22 mg/L. A blood triazolam concentration as high as 0.30 mg/L has been reported in an intoxication with a 0.10-g/dL blood-alcohol concentration (BAC) (6). Joynt (7) published blood data on four deaths due to triazolam alone. The blood concentrations ranged from 0.01 to 0.04 mg/L. Another nine deaths were identified with triazolam where the BAC exceeded 0.10 g/dL. The blood triazolam concentrations in these cases ranged from 0.01 to 0.053 mg/L. Kovesand Wells (8) reported three additional cases with a BACgreater than 0.10 g/dL; blood triazolam concentrations ranged from 0.022 to 0.065 mg/L. In the presented case, both heart blood and peripheral blood from the subclavian vein were collected. The ratio of heart blood to peripheral blood triazolam concentrations was 1.61. Dalpe-Scott et al. (10) reported ratios of heart blood to femoral blood triazolam concentrations in four postmortem cases. The average heart blood to femoral blood ratio was 2.8 with a range of 1.0 to 7.5. No actual blood concentrations were provided. Postmortem redistribution of benzodiazepines has not been widely reported. However, the volume of distribution of the basic drug triazolam (1.1-2.7 L/kg [2]), may suggest the possibility of postmortem redistribution. This is the first reported triazolam fatality where the major metabolite of triazolam, ~-OH-triazolam, was also measured. ~-OH-triazolam was not detected in either postmortem blood specimen at a limit of quantitation of 0.02 rag/L, but was detected in the liver, kidney, and urine. This is not surprising because the liver is the location of ~-OH-triazolam formation. Moreover, the urine was analyzed with and without glucuronide hydrolysis. As expected, most of the r was in the conjugated form. This is consistent with previously reported 53 Journal of Analytical Toxicology,Vol. 26, January/February2002 urine excretion studies involving triazolam (11). One unexpected finding was the high concentration of r detected in the kidney relative to the blood and liver. In a previously reported fatality due to alprazolam, a triazolobenzodiazepine like triazolam, no r was detected in the kidney (12). Because this is the only reported triazolam fatality where r is quantitated, it cannot be determined whether or not this observation is an outlier. Based on the investigation, autopsy, and toxicology findings, the medical examiner ruled that the cause of death was triazolam intoxication and the manner of death was suicide. References 1. Physicians' Desk Reference, 55th ed. Medical Economics Co., Montvale, NJ, 2001. 2. R.C. Baselt. Disposition of Toxic Drugs and Chemicals in Man, 5th ed. Chemical Toxicology Institute, Foster City, CA, 2000. 3. F.S. Eberts, Jr., Y. Philopoulos, L.M. Reineke, and R.W. Vliek. Triazolam disposition. Clin. Pharm. ?'her. 29:81-93 (1981). 4. T.S. Bal, B. Johnson, and E.A. Kilner. Three deaths involving triazolam-analytical aspects.J. Forensic ScL Soc. 29:119-123 (1989). 54 5. A. Steentoft and K. Worm. Cases of fatal triazolam poisoning. J. Forensic 5ci. 5oc. 33:45-48 (1993). 6. J.J.O'Dowd, RR Spragg, and RA. Routledge. Fatal triazolam poisoning. Br. Med. J. 297:1048 (1988). 7. B. Joynt. Triazolam blood concentration in forensic cases in Canada. J. Anal. Toxicol. 17:171-177 (1993). 8. G. Koves and J. Wells. The quantitation of triazolam in postmortem blood by gas chromatography/negative ion chemical ionization mass spectrometry. J. Anal ToxicoL 10:241-244 (1986). 9. K.K. Martin, J. Magluilo, Jr., and K.A. Moore. Solid phase extraction on the Zymark Rapid Trace Workstation for the screening of basic drugs. Presented at the 28th Society of Forensic Toxicologists Meeting, Albuquerque, NM, 1998 10. M. Dalpe-Scott, M. Degouffe, D. Garbutt, and M. Drost. A comparison of drug concentration in postmortem cardiac and peripheral blood in 320 cases. Can. 5oc. Forensic 5ci. J. 28:113-121 (1995). 11. A.D. Fraser,W. Bryan, and A.F. Isner. Urine screening for s-OH triazolam by FPIA and EIA and confirmation by GC/MS. J. Anal. Toxicol. 16:347-350 (1992). 12. A.J. Jenkins, B. Levine, J.L. Locke, and J.E. Smialek. A fatality due to alprazolam intoxication. J. Anal. Toxicol. 21:218-220 (1997). Manuscript received April 16, 2001; revision received June 26, 2001.