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Case Report
Abstract became the active ingredient in 1949 (2). The first reported
associated deaths from propylhexedrine toxicity were not re-
A death involving abuse of propylhexedrine and mitragynine is ported until 1974 (3).
reported. Propylhexedrine is a potent α-adrenergic Propylhexedrine is abused primarily by the intravenous
sympathomimetic amine found in nasal decongestant inhalers. route, although reports of oral ingestion have been described
The decedent was found dead in his living quarters with no signs of (4). Commonly referred to as “stove top speed”, propyl-
physical trauma. Analysis of his computer showed information on hexedrine can cause headache, tremor, chest pain, palpitation,
kratom, a plant that contains mitragynine, which produces opium- rapid respiration, dilated pupils, tachycardia, myocardial in-
like effects at high doses and stimulant effects at low doses, and a
farction, psychosis, nausea, pulmonary edema, and sudden
procedure to concentrate propylhexedrine from over-the-counter
inhalers. Toxicology results revealed the presence of 1.7 mg/L
death (1,3,4–7). One publication presented 15 cases of intra-
propylhexedrine and 0.39 mg/L mitragynine in his blood. Both venous propylhexedrine-related deaths, including 12 that died
drugs, as well as acetaminophen, morphine, and promethazine, as a result of propylhexedrine intoxication. Nine of the 12
were detected in the urine. Quantitative results were achieved by showed toxic effects with anatomical indications of right ven-
gas chromatography–mass spectrometry monitoring selected ions tricular hypertrophy and pulmonary hypertension at autopsy
for the propylhexedrine heptafluorobutyryl derivative. Liquid (1).
chromatography–tandem mass spectrometry in multiple reactions Mitragynine is the main alkaloid found in the leaves of Mi-
monitoring mode was used to obtain quantitative results for tragyna speciosa, a plant that is known as kratom in Thailand
mitragynine. The cause of death was ruled propylhexedrine and biak-biak in Malaysia (8,9). Kratom contains many other
toxicity, and the manner of death was ruled accidental. indole alkaloids that are structurally related to mitragynine, in-
Mitragynine may have contributed as well, but as there are no
cluding mitraphylline, speciogynine, speciociliatine, paynan-
published data for drug concentrations, the medical examiner did
not include mitragynine toxicity in the cause of death. This is the
theine, ajmalicine, and 7-hydroxymitragynine (10,11).
first known publication of a case report involving propylhexedrine Mitragynine is the major component of Mitragyna speciosa
and mitragynine. with a reported concentration as high as 6% by weight of the
dried plant material and as much as 66% of the crude base
(10,11). Mitragynine is ingested orally by chewing fresh leaves
or by drinking a tea brewed with the substance (12).
Mitragynine is used for its opium-like effects at high doses.
Introduction
At low doses, it has stimulant-like effects similar to the coca
plant. Many laborers in Asia use mitragynine to combat fa-
Propylhexedrine is the active ingredient in over-the-counter
nasal decongestants Benzedrex® and Dristan®. It is a potent α-
tigue (10). It can also be used for opiate withdrawal, fever re-
duction, analgesia, diarrhea, coughing, and hypertension
adrenergic sympathomimetic amine that acts as a local vaso- (8,13). Mitragynine is reported to act on the µ-opioid receptors
constrictor with one-twelfth the central nervous system stim- to elicit analgesic effects (14). There are no well-defined studies
ulant effects and less than one-eighth of the pressor effects of to show toxicity of mitragynine. It is currently not listed as a
amphetamine (1). After reports of amphetamine inhalers being scheduled drug in the United States. Mitragynine is not the
dismantled to extract the drug for abuse, propylhexedrine only active compound present in Mitragyna speciosa; 7-
hydroxymitragynine is also active and reported to have more
potent analgesic effects than mitragynine. However, it is only
* Disclaimer: The opinions or assertions contained herein are the private views of the authors 0.04% by weight in the plant material (11,15). Other compo-
and are not to be construed as official or as reflecting the views of the Department of Defense,
the Army, Navy, or Air Force.
nents of the plant and some metabolites are reported to be ac-
† Author to whom correspondence should be addressed. E-mail: blevi001@umaryland.edu. tive as well (10,13).
54 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission.
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Mitragynine instrumental analysis sured for the validation of propylhexedrine and mitragynine in
The LC–MS–MS analysis of mitragynine was performed human blood: selectivity, specificity, linearity, limit of quanti-
using an Agilent 1100 series HPLC system (Palo Alto, CA) cou- tation (LOQ), limit of detection (LOD), upper limit of linearity
pled with an Applied Biosystems/MDS SCIEX 3200 QTRAP (ULOL), within- and between-day precision, accuracy, and ex-
(Foster City, CA) equipped with a Turbo V™ source. Analyst 1.5 traction recovery. Ion suppression effects were also evaluated
software was used for data acquisition and analysis. for the LC–MS–MS method.
Chromatographic separation was performed on an Agilent Selectivity was examined by extracting and analyzing six
Zorbax XDB C18 column (4.6 × 75 mm, 3.5 µm, Palo Alto, CA) different and separate sources of negative matrices. This was
with a Mac-Mod Analytical Halo™ C18 (4.6 × 20 mm, 2.7 µm, done to evaluate any significant, extraneous peaks that may in-
Chadds Ford, PA) used as a guard column. The column com- terfere with the analysis at the retention time of the analytes.
partment was maintained at 40°C, and the injection volume Specificity was evaluated by analyzing positive quality control
was set at 10 µL. The mobile phase was set at a constant flow samples which contained 87 frequently encountered com-
of 500 µL/min and consisted of (A) 0.1% formic acid in deion- pounds for possible interference with the assay.
ized water and (B) acetonitrile. A gradient elution was used Linearity was assessed by the analysis of a multipoint cali-
and is listed as follows: pre-injection equilibration with 70% bration curve (linear regression) on separate days. The LOQ
A for 4.0 min, hold at 70% A for 1.0 min after injection, ramp was determined by the lowest concentration which meets all
to 45% A over 4.0 min, hold until 7.0 min, ramp to 35% A at the criteria for confirmation and is calculated within ±20% of
8.0 min, return A to 70% by 8.50 min, for a total run time of the theoretical spiked mean. The LOD was determined to be
8.5 min. Detailed HPLC gradient conditions are shown in the lowest concentration that meets all the criteria for confir-
Table I. mation but the calculated concentration is greater than ±20%
The MS was operated in positive electrospray ionization of the theoretical spiked mean. The ULOL was determined by
mode (+ESI). The analysis of mitragynine and proadifen (ISTD) the highest concentration which meets all the criteria for con-
was operated in multiple reactions monitoring (MRM) acqui- firmation and the calculated concentration was within ±20%
sition mode. Two MRM transitions were monitored for mi- of the theoretical spiked mean.
tragynine (m/z 399.3/174.1* and m/z 399.3/226.3) and one Within-day precision was measured by the analysis of five
transition for the ISTD (m/z 354.3/91.1*) (* denotes quantita- aliquots of three controls at different concentrations in a
tion ion). single extraction. Between-day precision was measured with
The source dependent parameters for the MS–MS analysis of the analysis of one aliquot of three controls over five separate
mitragynine were determined by the flow injection analysis. extractions. The acceptable value of precision, expressed as
Multiple injections of a 20 mg/L mitragynine standard were the coefficient of variation (CV), for each within-day and be-
made, bypassing the analytical column while varying different tween-day assays must be < 15%. Accuracy, defined as per-
source parameters with each sequential injection. The opti- cent difference from the theoretical spiked concentration
mized source-dependent parameters for the analysis were as (% diff), was determined for each of the three controls over
follows: GS1 gas (nebulizer) was set to 50 psi, GS2 gas (turbo)
was set to 60 psi, CUR (curtain gas) was set to 25 psi, TIS (Tur- Table I. Gradient Elution Timetable*
boIonSpray® voltage) was set to 1500 V, and the source tem-
perature was set at 350°C. Time Flow Rate
The compound dependent parameters for the MS–MS anal- Step (min) (µL/min) %A %B
ysis of mitragynine were determined by direct infusion. An in-
tegrated infusion pump delivered a 10 mg/L standard solution 0 4.0 500 70 30
at a constant flow (10 µL/min) directly into the TIS source. The 1 1.0 500 70 30
auto-optimization process determines the optimal parameters 2 4.0 500 45 55
3 7.0 500 45 55
for each MRM transition. The following parameters were opti-
4 8.0 500 35 65
mized during the process: DP (declustering potential), EP (en- 5 8.5 500 70 30
trance potential), CEP (collision cell entrance potential), CE
(collision energy), and CXP (collision cell exit potential). Table * (A) 0.1% formic acid in deionized water; (B) acetonitrile.
II lists the optimal compound dependent parameter for mi-
tragynine and internal standard.
Identification of mitragynine in the
Table II. Optimal Compound-Dependent MS–MS Parameters
case specimens was based on the ion
ratio of MRM2/MRM1 transitions being
Compound-Dependent Parameter
within 20% of the average ion ratios and MRM Transition
the relative retention time being within Compound (m/z/m/z) DP EP CEP CE CXP
±2% of the averages measured from the
calibrators. Mitragynine 399.3/174.1 60.0 4.0 25.0 40.0 4.0
399.3/226.3 60.0 6.0 25.0 35.0 2.0
Method validation Proadifen (ISTD) 354.3/91.1 37.0 10.0 24.0 50.0 3.0
The following parameters were mea-
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each extraction. Each control concentration within ±20% of frequently encountered compounds for propylhexedrine or
the theoretical spiked concentration was considered accept- mitragynine. No significant ion suppression or enhancement
able. was detected with either matrices (blood and urine) or the
Extraction recovery studies were performed by comparing mobile phase. The precision, accuracy, and linearity all meet
unextracted standards to extracted blood samples where the the acceptable criteria for validation. The validation data, in-
drug was added post- and pre-extraction. The study was per- cluding linearity, LOQ, LOD, ULOL, precision, accuracy, and
formed at several different concentrations over the linear extraction recovery, are summarized in Table III.
range. The extraction recovery was calculated as the differ-
ence between the samples where the standards were added Case results
post-extraction versus pre-extraction. The ion suppression or No ethanol (cutoff 0.02 g/dL) or other volatile substances
enhancement was determined to be the signal difference, (cutoff 0.001 g/dL) were detected in the decedent’s blood and
positive or negative, between the extracted blank matrix, vitreous fluid. Urine immunoassay screening produced posi-
the mobile phase, and the unextracted infused mitragynine tive results for the Roche (Indianapolis, IN) Abuscreen Online
standard. Amphetamines and Opiates assays. Confirmation testing for
amphetamines failed to identify amphetamine, methampheta-
mine, phenylpropanolamine, pseudoephedrine, ephedrine,
methylenedioxyamphetamine, and methylenedioxymetham-
Results phetamine at an LOQ of 0.05 mg/L in urine. Opiate confir-
mation testing showed presence of morphine in the urine, not
Method validation in the blood and negative for codeine, hydrocodone, hydro-
The validation data met all acceptable criteria for method morphone, oxycodone, and oxymorphone at an LOQ of 0.05
validation. No interferences, excessive background, or en- mg/L for blood and urine. 6-Acetylmorphine was negative by
dogenous peaks were detected in six separate sources of nega- immunoassay at a 10 ng/mL cutoff. A full-scan GC–MS base
tive blood and urine. No extraneous interfering peaks or ions screen detected promethazine, propylhexedrine, and mitrag-
were detected from the quality control samples containing 87 ynine in his urine. The FPIA for acetaminophen was positive
in the urine and confirmed by color test. No other thera-
peutic or abused drugs were detected. Table IV provides quan-
Table III. Method Validation Data for Propylhexedrine
and Mitragynine in Blood titative results in the blood; Table V represents the tissue
distribution for propylhexedrine and mitragynine in speci-
Mitragynine Propylhexedrine mens received.
Figure 1.Total ion chromatogram of the decedent’s urine including product ion scans for 17-O-desmethyldehydromitragynine (A), 7-hydroxymitragynine (B),
9-O-desmethylmitragynine (C), 16-carboxymitragynine (D), mitragynine (E), speciogynine (F), speciociliatine (G), unidentified peak (H), and paynantheine (I).
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