A Fatality Due To Alprazolam Intoxication - Journal of Analytical ...

] Case Report
A Fatality Due To Alprazolam Intoxication
Amanda J. Jenkins, Barry Levine*, J. taron Locke, and John E. Smialek
Office of the Chief Medical Examiner, State of Maryland, Baltimore, Maryland
Abstract
Alprazolam is one of the most widely prescribed benzodiazepines
in the United States. It is generally considered a safe and effective
drug for the treatment of anxiety disorders and panic attacks.
Few overdoses that are due to the sole ingestion of alprazolam
have been reported. This paper documents a fatality due to
alprazolam intoxication and describes the distribution of
alprazolam and an active metabolite, (x-hydroxyalprazolam, in
tissues obtained at autopsy. Qualitative identification of the drugs
was achieved by full-scan gas chromatography-mass spectrometry,
and quantitative analysis was performed by high-performance
liquid chromatography. High concenlrations of alprazolam were
found in all specimens analyzed, but the metabolite was detected
only in subclavian blood, urine, bile, and liver. A postmortem heart
blood alprazolam concentration of 2.1 mg/t is the highest
reported in the literature to date.
Introduction
Alprazolam (8-chloro- 1-methyl-6-phenyl-4H-s-triazolo
[4,3-c~] [ 1,4] benzodiazepine, Xanax | is one of the most widely
prescribed benzodiazepines in the United States. It is com-
monly prescribed for the treatment of anxiety and depression
and for sedation (1). It is an effective anxiolytic agent at doses
of 0.75-4 mg daily, and doses of 6-9 rng per day are prescribed
to treat panic attacks and panic disorders (2). Its precise mech-
anism of action is unknown, but, presumably, it acts in a
manner similar to other benzodiazepines by potentiating
GABA. Following oral administration, it is rapidly absorbed
with peak plasma concentrations occurring 1-2 h after inges-
tion. McCormick et al. (3) reported steady-state serum con-
centrations of 25-55 ng/mL of alprazolam in six individuals
after chronic daily oral administration of 1.5-6 rng. Dose-
related increases in steady-state plasma concentrations were
reported with average concentrations reaching 102 ng/mL
after doses of 9 rag/day (4).
"Addres ~, R)r c orresponden(e: Dr. Barry Levine, Offi( e (ff the Chief Medical Examiner, 111 Penn
Stret4, Baltimore, MD 21201.
Journal of Analytical Toxicology, Vol. 21, May/June 1997
Alprazolam has a short duration of action with an average
plasma half-life of 11 h (1). It is extensively metabolized by
oxidation and conjugation with only 20% of the parent drug
appearing unchanged in urine (5). The major metabolites are
~-hydroxyalprazolam, 4-hydroxyalprazolam, r
alprazolam, and 3-hydroxy-5-methyltriazolyl chlorobenzo-
phenone (HMTBP). r and 4-hydroxy-
alprazolam are pharmacologically active with approximately 66
and 19% of the potency of alprazolam, respectively (5). Smith
and Kroboth (6) reported that unconjugated forms of these two
metabolites were detected at plasma concentrations of less
than 10% of the parent drug concentration even after chronic
dosing, and, therefore, their contribution to pharmacological
effects may be minimal in therapeutic cases.
Adverse reactions to alprazolam are typically observed at the
beginning of therapy and diminish under continued treatment.
The most common effects reported are drowsiness and fatigue.
Other adverse reactions include confusion, headache, nausea
and vomiting, tachycardia, hypotension, and blurred vision (2).
Abrupt cessation of chronic alprazolam therapy has resulted in
withdrawal symptoms, the most important being seizure
activity (2). However, alprazolam is generally considered a safe
and effective drug with a high therapeutic index. Few over-
doses attributable to alprazolam have been reported, and even
fewer fatalities due to alprazolam intoxication without the com-
bination of other drugs are documented. In this study, we report
the tissue distribution of alprazolam and c~-hydroxyalprazolam
in a death due solely to the ingestion of alprazolam.
Case History
The deceased was a 44-year-old white female with a history
of psychiatric problems. She was found dead in bed at her res-
idence. Her current medications, which were found at the
scene, included alprazolam, fluoxetine, phenytoin, lorazepam,
and venlafaxine. Although there was a history of suicidal
ideation, there was no suicide note.
At autopsy, external examination showed a well-nourished,
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Journal of Analytical Toxicology, Vol. 21, May/]urge 1997
well-developed female. Internal examination was unremark-
able except for 60 mL of a white granular substance and 40 mL
of a green fluid in the stomach. Microscopic findings demon-
strated mild emphysematous changes in the lungs with intra-
alveolar hemorrhages and focal mild pulmonary edema. Spec-
imens collected for toxicological analysis were stored in sterile
polypropylene containers without preservative and were frozen
at -20~ until assayed.
Methods
Materials
Alprazolam and (z-hydroxyalprazolam were purchased from
Sigma Chemical (St. Louis, MO). Chlordiazepoxide was ob-
tained from Alltech Applied Science Labs (State College, PA).
Analytical-grade sodium borate was purchased from Mallinck-
rodt (St. Louis, MO), and a saturated solution was prepared for
use as a buffer. Reagent-grade methylene chloride, methanol,
and diethylamine were obtained from Fisher Scientific (Fair
Lawn, N J). Solid-phase extraction columns (Chem Elut TM)
were purchased from Varian (Harbor City, CA).
Procedure
Alprazolam and e(-hydroxyalprazolam analytical standards
were prepared in methanol at a concentration of 100 mg/L.
Chlordiazepoxide (100 rag/L), the internal standard, was also
prepared in methanol. Internal standard (50 IJL) was added to
2 mL of blank blood or case specimen and 2 mL of buffer
solution. Standard curves for alprazolam and (z-hydroxyalpra-
zolam were prepared at the following concentrations: 0, 0.5,
1.0, 2.5, and 5 mg/L. Homogenates of tissue samples were pre-
pared by adding 1 g tissue to 4 mL distilled water. Samples were
vortex mixed and applied to solid-phase extraction columns.
The tubes were rinsed with 1 mL buffer, which was then applied
to the columns. Drugs were eluted from the column with
2 x 7 mL methylene chloride. The eluent was evaporated to
dryness, and the residue was reconstituted with 400 1JL
methanol. An aliquot was injected onto the high-performance
liquid chromatograph (HPLC) for quantitative analysis. The
assay was linear from 0.2 to 5.0 mg/L with a limit of sensitivity
of 0.2 mg/L. Drug identification was achieved by full-scan gas
Table I. Tissue Distribution of Alprazolam and
c~-Hydroxyalprazolam in Overdose Death
Specimen Alprazolam (x-Hydroxyalprazolam
Heart blood (mg/L) 2.1 Not detected
Subclavian blood (mg/L) 2.3 0.12
Urine (mg/L) 1.0 1.4
Bile (mg/L) 2.8 1.3
Vitreous humor (rag/L) 0.58 Not detected
Liver (mg/kg) 9,2 0.83
Kidney (mg/kg) 3.8 Not detected
Stomach contents 13 mg in 110 mL Not detected
chromatographic-mass spectrometric (GC-MS) analysis and
comparison with an authentic standard.
HPLC instrument conditions
A Waters (Milford, MA) 501 HPLC pump interfaced with a
Waters 715 Ultra Wisp sample processor, a Waters 490E pro-
grammable multiwavelength detector, and a Hewlett-Packard
(Palo Alto, CA) integrator model 3394A were used for the quan-
titative determination of alprazolam and c(-hydroxyalprazolam.
A Beckman (San Ramon, CA) ODS column (15 cmx 4.6-ram
i.d., 5-]Jm particle size) was heated to 30~ The detector wave-
length was 240 nm. The mobile phase consisted of methanol
and water (60:40) with 1% diethylamine (pH 6) and a flow
rate of 1.5 mL/min.
Results and Discussion
A comprehensive toxicological screen was performed on the
heart blood and urine specimens obtained in this case. This
included ethanol and volatile analysis by headspace GC,
radioimmunoassay for morphine, a liquid-liquid extraction
followed by GC-nitrogen-phosphorus detection (NPD) for basic
drugs, and solid-phase extraction with GC-NPD for acidic or
neutral drugs. In addition, color tests were performed for acetaminophen,
aspirin, and ethchlorvynol. The urine was positive
for alprazolam, chlorpheniramine, dextromethorphan, and
doxylarnine. The heart blood was positive for alprazolam only.
Using the normal base extraction procedure therapeutic concentrations
of alprazolam are usually undetected because the
limit of detection is 0.1 mg/L. However, in this case, alprazolam
was easily detected by GC-NPD after base extraction was performed
and a full-scan electron impact mass spectrum was
obtained.
Quantitation of alprazolam and c(-hydroxyalprazolam in biological
specimens was performed by reversed-phase HPLC.
This procedure is commonly used for the analysis of benzodiazepines
(7-9). In this assay, the order of elution was
~-hydroxyalprazolam, alprazolam, and the internal standard.
The relative retention times with respect to internal standard
were 0.70 and 0.84 rain for (x-hydroxyalprazolam and alprazolam,
respectively. Chlordiazepoxide was chosen as the
internal standard to enable baseline resolution of the three
drugs while optimizing analysis time.
The quantitative results obtained in this case are shown in
Table I. Alprazolam was detected in all specimens analyzed.
Concentrations of alprazolam >2 mg/L were measured in both
heart and subclavian blood specimens. There was good agreement
in the alprazolam concentration between the two blood
specimens. The alprazolam concentration in liver was approximately
2.4 times the concentration in kidney. The concentration
of parent drug measured in bile was approximately three
times the concentration determined in urine, which suggested
that this specimen would be useful for an initial screening for
those assays that are not targeted to drug metabolites. This case
also showed a readily detectable concentration of alprazolam in
the vitreous humor. Given the relative protection of the vit-
219

eous from putrefactive processes, drug screening may be per-
formed on this tissue in the absence of other appropriate spec-
imens. The metabolite r was only detected
in the subclavian blood, urine, bile, and liver specimens.
or was present at 5.2% of the concentration
of parent drug in the blood, which agrees with the work of
Smith and Kroboth (6). Therefore, even at fatal concentra-
tions, the relative contribution of r to tox-
icity may be minimal.
The tissue distribution of alprazolam described in this report
differs from that of some other benzodiazepines noted in
fatalities that are due to intoxication. Cardauns and Iffland
(10) described the tissue distribution of diazepam and its active
metabolite, nordiazepam, in an intoxication. The concentration
of the parent drug was 10 times greater in blood than urine
compared with two times greater concentration in the alpra-
zolam case described in this report. Furthermore, the con-
centration of diazepam in the kidney was 20 times less than the
concentration in the liver. In this case, the concentration of
alprazolam in the kidney was only 2.4 times less than its con-
centration in the liver. Cardauns and Iffland (10) found rela-
tively low concentrations of nordiazepam in the tissues ana-
lyzed; the highest concentration was reported in the urine.
This was similar to the distribution of cz-hydroxyalprazolam
reported in this study.
In a fatality attributed solely to chlordiazepoxide intoxication
(1), the concentration of the parent drug in the blood was
about three times the concentration in urine. Concentrations
in the liver and kidney were approximately equivalent, and
the highest concentration of drug in the specimens analyzed
was detected in the bile. Metabolite concentrations were not
reported in this case.
There are only two reports of fatal intoxications that were
due to the ingestion of alprazolam documented in the litera-
ture. Edinboro and Backer (7) reported a blood alprazolam
concentration of 0.177 mg/L in an antemortem hospital
admission specimen from a depressed and suicidal woman. In
another study, Stafford et al. (11) reported a postmortem blood
alprazolam concentration of 0.122 mg/L in an acute alpra-
zolam intoxication with concomitant ingestion of ethanol
(postmortem blood alcohol concentration = 0.15 g/dL). If the
therapeutic range is considered to be 0.025-0.102 mg/L,
depending upon low- or high-dose therapy, the alprazolam
concentrations determined in both of the previous fatalities
were 1.2-7.1 times therapeutic levels. In comparison, the mean
blood concentration determined in this case was 22-90 times
greater than the therapeutic range. These concentrations are
the highest reported in the literature to date.
220
Conclusion
Journal of Analytical Toxicology, Vol. 21, May/June 1997
Following the completion of the investigation and the eval-
uation of the autopsy and toxicology findings, the medical
examiner ruled the cause of death as acute alprazolam intoxi-
cation. Given the past psychiatric history of suicidal ideation
and the alprazolam concentration in the blood, the manner of
death was ruled suicide.
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Manuscript received July 29, 1996;
revision received October 25, 1996.