Journal of Analytical Toxicology 2014;38:615 617

doi:10.1093/jat/bku073

Case Report

Suspected Impaired Driving Case Involving a-Pyrrolidinovalerophenone, Methylone

and Ethylone
Justin L. Knoy*, Brianna L. Peterson and Fiona J. Couper
Toxicology Laboratory Division, Washington State Patrol, 2203 Airport Way S., Suite 360, Seattle, WA 98134, USA
*Author to whom correspondence should be addressed. Email: justin.knoy@wsp.wa.gov

This is the first reported case of a-pyrrolidinovalerophenone (a-PVP),

methylone and ethylone in a suspected impaired driving case in the
state of Washington. An initial traffic stop by law enforcement was
made of a driver due to poor navigation of the roadway. The drug recognition expert (DRE) officer observed slurred speech, bloodshot watery eyes, dilated pupils, involuntary muscle movements and an
elevated pulse and blood pressure. The DRE deduced that the driver
was likely under the influence of central nervous system (CNS) stimulants, specifically bath salts. Routine testing of the blood did not
reveal the presence of alcohol or common drugs of abuse. Upon further review of the officers report and the unconfirmed identification
of a-PVP, blood was sent to NMS Labs in Willow Grove, PA, USA for
bath salts and stimulant designer drugs testing. Analysis was conducted by liquid chromatography time-of-flight mass spectrometry
with the following results: 63 ng/mL a-PVP, 6.1 ng/mL methylone
and positive for ethylone. These results are consistent with the
DRE opinion of driving performance being impaired by a CNS
stimulant.

Introduction
The increase in use of designer drugs known as bath salts has
proven frustrating to both law enforcement agencies and forensic laboratories alike. There are a multitude of difculties laboratories face in implementing analytical methods that can
effectively and efciently identify and quantify these compounds.
Pure reference standards of these drugs must rst be acquired,
which can prove both challenging and expensive. Time and
resources used to complete casework must be redirected to developing these methods within budget constraints. In the meantime, the drugs of interest often become replaced with various
chemical analogs when the current drugs on the market become
scheduled, requiring laboratories to start the process all over
again. It is for these reasons that when cases submitted to the
toxicology laboratory of the Washington State Patrol (WSP)
have case histories with obvious observations of impairment, indications that a bath salt has likely been abused, and no other
drugs detected, the specimen is sent out for testing to a reference laboratory that has the time and resources to develop appropriate conrmation methods for this rapidly evolving group
of drugs.
This is a case report of an individual who was stopped for
poor navigation of the roadway and arrested for suspicion of driving under the inuence (DUI) of drugs. Three synthetic cathinones, which are often referred to generically as bath salts,
were detected in the subjects blood. The compounds were
a-pyrrolidinovalerophenone (a-PVP), ethylone and methylone.
The subjects driving behavior, performance on eld sobriety
tests and other clinical signs and symptoms are summarized.

Case report
Arrest
A 34-year-old male was stopped by a law enforcement ofcer for
improper lane travel and stopping in the wrong place. He was reported to have stopped 10 feet before a stop sign for no apparent reason. He then drove forward and stopped again. The driver
entered the oncoming lane of travel upon turning onto the intersecting highway before quickly moving into the correct lane. The
arresting ofcer observed the vehicle cross the center line three
times in the span of 1.5 miles before initiating a trafc stop. The
driver was observed to appear disoriented and confused. He was
reported to have slurred speech, bloodshot watery eyes and difculty dividing his attention. A portable breath test was administered with the result of 0.000. The ofcer then conducted
standardized eld sobriety tests (SFSTs), to which the driver presented the following: two of six clues on the horizontal gaze nystagmus (HGN) test, seven of eight clues on the walk-and-turn
(WAT) test and four of four clues on the one-leg stand (OLS)
test. The Romberg balance test was also conducted with the following results: extreme eyelid tremors, swaying and an inability
to stand still. The driver admitted to having narcolepsy and taking
the prescription medicines Klonopinw and Depakotew. At this
point, the driver was placed under arrest for DUI.

Drug Recognition Expert Evaluation

A drug recognition expert (DRE) conducted an evaluation of the
driver. Initial observation showed that the individual was pale,
had bloodshot watery eyes, a drowsy appearance and uctuated
between being calm and agitated. During the psychophysical
tests, the subject was observed to have a fast internal clock
when performing the Romberg balance test, estimating the passage of 30 s in 24 s. During the test, the subject swayed and it was
noted that he had severe eyelid tremors and upper body muscle
tremors. During the WAT test, he became agitated during the instructional phase and exhibited six of eight clues including inability to keep balance, stopped walking, stepped off the line,
missed heel to toe, raised his arms for balance and hopped during
the turn. During the OLS test, he was observed to sway, use his
arms for balance, hop and put his foot down twice prior to the
conclusion of the test. The subject had difculty performing the
nger-to-nose test. During this test, an individual is instructed to
touch the tip of his nose with the tip of his nger. The subject
used the pad of his nger rather than the tip on several instances
and touched his nostrils rather than the tip of his nose multiple
times.
Both the HGN test and the vertical gaze nystagmus (VGN) test
were administered. The subject presented two of six clues on the
HGN test; however, the test was unable to be completed because
the subject repeatedly looked away from the stimulus while the

# The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com

ofcer checked for sustained and distinct nystagmus at maximum deviation. VGN was not detected. Lack of convergence
was found to be present.
The DRE then checked the individuals pupil size and reaction to
light. The pupils were found to be dilated in all light conditions:
7 mm in diameter in room light (normal: 2.5 5 mm), 9 mm in
total darkness (normal: 58.5 mm) and 7.5 mm in direct light (normal: 24.5 mm). Overall reaction to light was noted to be slow.
Subsequent to the eye examination, the DRE checked the individuals vital signs. The pulse was checked at three separate times
over the course of 30 min with readings of 90, 106 and 80 beats
per minute (BPM). When compared with the average range used
for DRE purposes of 6090 BPM, it is seen that there is a somewhat elevated pulse. The subject had an elevated systolic blood
pressure of 150/82 mmHg (normal: 120140/7090). The subjects body temperature was measured and found to be 98.68F,
which was within normal range (98.6 + 1.0). Muscle tone was determined to be normal. In addition to these ndings, the DRE observed raised taste buds and thick white saliva on the tongue.
The subjects statements were also recorded. He admitted to
using Kratom, Prozac w, Saphrisw, Risperidonew, Depakotew,
Nuvigilw and Klonopinw. In addition to these admissions, a
white powdery substance was found in possession of the subject
who claimed it contained the following: Energy Powder, Blast
Off, Fusion, Fast Forward, Nalt and B-12. However, this was not
conrmed as the powder was not analyzed by our laboratory.
Upon completion of the examination, it was the opinion of the
DRE that the subject was under the inuence of central nervous
system (CNS) stimulants, specically Kratom and bath salts.
Blood was drawn 2 h and 9 min after the initial arrest/stop.

Analysis
Two blood samples were submitted to the WSP toxicology laboratory for testing. Samples are stored at all times in refrigerators
upon sample receipt except for testing purposes. Routing testing
consisted of an initial analysis for volatiles by headspace gas chromatography (GC) followed by an immunoassay screen utilizing
enzyme multiplied immunoassay technique (EMIT) for amphetamines, barbiturates, benzodiazepines, cannabinoids, cocaine
metabolite, methadone, opiates, phencyclidine and tricyclic antidepressants. All results were negative. An alkaline drug screen by
way of liquid liquid extraction was conducted. Then, 1.0 mL of
reference mix, control or case sample was added to 1.0 mL of pH
9 borate buffer along with the addition of metycaine (0.5 mg, internal standard). After vortexing, 3.0 mL of n-butyl chloride was
then added followed by rotation for 20 min on a rotary mixer.
Samples were then centrifuged for 10 min at 3,500 rpm. The organic layer was transferred to a clean tube, followed by the addition of 200 mL of 3 N HCl. The samples were again placed on a
rotary mixer for 5 min, and then centrifuged for 5 min at
2,000 rpm. The organic layer was aspirated to waste, while
100 mL of both saturated ammonium carbonate and concentrated ammonium hydroxide and 150 mL of chloroform were added
to each tube. After vortexing, samples were again centrifuged at
2,000 rpm for 5 min. The chloroform layer was then transferred
to two autosampler vials with inserts. In order to achieve separation and identify any compounds, 3 mL of each extract was injected onto an Agilent 6890/5973 GC MS system. A 30-m, 0.25-mm
616 Knoy et al.

diameter, 0.25-mm lm thickness HP5-MS column was used. The

front inlet temperature was 2508C, and a pulsed splitless mode
was utilized. The initial oven temperature was 908C with a
ramp of 158C/min to 1808C, followed by a ramp of 108C/min
to 3008C, and then held for 10 min. The MS transfer line was
set to 2808C. The MS was set in scan mode with a solvent delay
of 3 min. The unconrmed spectral match of a-PVP was found
utilizing an uncertied reference MS library. No other drugs
were detected above reporting limits from either an approved
MS library or otherwise. Additional testing using highperformance liquid chromatography tandem mass spectrometry for analysis of benzodiazepines, including clonazepam and
7-aminoclonazepam [limit of quantitation (LOQ): 0.01 mg/L],
was performed, with no drugs detected. Even though the initial
EMIT screen was negative for benzodiazepines, this testing was
performed because of the admission of Klonopinw and the
knowledge that low concentrations of this drug do not always
produce a positive response from the immunoassay. Analysis
for valproic acid was performed using GC with ame ionization
detection, with no drug detected (LOQ 10 mg/L). The subject
had stated use of Depakotew, and this testing was performed due
to this admission.
In order to conrm the presence of a-PVP and any other compounds classied as bath salts or designer stimulant drugs, the
sample was sent to NMS Labs for analysis. Personal communications (D. Papsun, 10 March 2014) with an analyst at NMS Labs
provided the following information regarding their protocol for
conrming these compounds. Preparation of the sample at NMS
Labs begins by transferring 500 mL of blanks, calibrators, controls
and specimens to tubes. Twenty-ve microliters of working
internal standard (d5-PCP, d5-MDA, d4-norketamine HCl,
d3-atropine, d5-MDMA and d4-ketamine HCl; 200 ng/mL) were
then added to each tube followed by vortexing. After that 1 mL
of 0.1 M pH 10.4 borax buffer was added, followed by 3.0 mL of
n-butyl chloride ethyl acetate (70 : 30). The samples were then
capped, allowed to rotate for 1015 min, centrifuged for 10 min
and the upper organic layer was transferred to new tubes. The
organic layer was then evaporated to dryness under nitrogen at
408C for 15 min. The samples were reconstituted with 200 mL
of reconstitution solution [9 : 1; mobile phase A (0.05% formic
acid in 5 mM ammonium formate) to mobile phase B (0.05% formic acid)]. After vortexing, the samples were transferred to
automated liquid sampler vials for high-performance liquid
chromatography time-of-ight mass spectrometry (HPLC
TOF/MS) analysis. A 10-min chromatography gradient was
implemented with the mobile phases of ammonium formate
and formic acid using positive mode electrospray. The LC
TOF/MS is an Agilent Jet Stream 6230 with Wellplate Sampler
G1367D, Binary Pump G1312B and Thermostatted Column
Compartment G1316B. Pumps were programed to deliver an increasing gradient of mobile phase B against an aqueous mobile
phase A. The solvent composition changed over 10 min from
95% mobile phase A : 5% mobile phase B to 5% mobile phase A :
95% mobile phase B. The column used for separation was a
Zorbax Eclipse Plus C18 Rapid Resolution HT, 3.0  100 mm,
1.8 mm. Injection volume was 5 mL with automated needle washes in between. The mass analyzer scanned in different time segments from 80 to 1,700 m/z. The following results were
obtained: 63 ng/mL a-PVP, 6.1 ng/mL methylone and positive
for ethylone (reporting limit10 ng/mL). Mitragynine and

7-hydroxymitragynine (Kratom) are additional compounds that

can be conrmed by this analysis, but were not detected (reporting limit10 ng/mL).

Discussion
Bath salts consist of a growing class of synthetic cathinones that
have stimulant and hallucinogenic properties. Methylone
(3,4-methylenedioxy-N-methylcathinone) and ethylone (3,4methylenedioxy-N-ethylcathinone) belong to the subset
identied as b-ketone amphetamines. They are analogs of 3,4dimethoxymethamphetamine (MDMA) and 3,4-dimethoxyethylamine (MDEA), respectively, as they differ only by the presence
of a ketone at the b-carbon (Figure 1).
These drugs are known to inhibit the reuptake of dopamine,
norepinephrine and serotonin, or conversely increase the release
of these neurotransmitters from intracellular stores (1 3). a-PVP
belongs to the subset of pyrrolidinophenones, structurally similar
to methylenedioxypyrovalerone (MDPV). When compared with
cocaine and amphetamine, both MDPV and a-PVP have been
shown to be much more potent inhibitors of dopamine and norepinephrine reuptake (4) (Figure 2).

It is due to this mechanism of action that synthetic cathinones

are considered CNS stimulants. Common subjective effects include increased energy, euphoria, empathy, openness and increased libido (5, 6). Reported adverse effects include
tachycardia, confusion, agitation, insomnia, aggression, hallucinations and hypertension (2, 6, 7).
This is the rst reported instance of a-PVP and ethylone in the
Washington State from either a suspected impaired driver or
death investigation. Methylone has previously been reported,
but not in combination with these other two compounds. The
observations of the arresting ofcer and the DRE are consistent
with what one might expect from an individual under the inuence of these compounds. In addition to poor navigation of the
roadway, the driver appeared confused, disoriented and agitated
at times. The subject was also observed to have involuntary muscle movements at various times during the performance of the
SFSTs, both by the arresting ofcer, and again during the DRE
evaluation. It has been observed that when a-PVP has been administered to mice, there is a signicant increase in locomotor
activity due to increased extracellular levels of dopamine (1, 4).
This phenomenon was noted to have a quicker time of onset and
to occur at a more intense rate. The clinical indicators present in
this case of tachycardia, mydriasis and body tremors are also consistent with the known effects of CNS stimulants and of these
compounds specically.
Due to the thorough account by the DRE involved, this case
presents a unique opportunity to understand the psychophysical
effects of a-PVP, methylone and ethylone on a subject in a real
world driving situation. The analytical ndings further the observations of ofcers involved and are consistent with the DREs
opinion of an individuals driving performance being adversely affected by a CNS stimulant. This case report illustrates how invaluable the observations of a DRE can be to a forensic toxicologist
when analyzing samples submitted where an arresting ofcer
observes strong indications of impairment, but alcohol is ruled
out as the causing agent.

References
Figure 1. Structures of methylone, MDMA, ethylone and MDEA.

Figure 2. Structures of a-PVP and MDPV.

1. Kaizaki, A., Tanaka, S., Numazawa, S. (2014) New recreational drug

1-phenyl-2-(1-pyrrolidinyl)-1-pentanone (alpha-PVP) activates central
nervous system via dopaminergic neuron. The Journal of
Toxicological Sciences, 39, 1 6.
2. McIntyre, I.M., Hamm, C.E., Aldridge, L., Nelson, C.L. (2013) Acute
methylone intoxication in an accidental drowninga case report.
Forensic Science International, 231, e1 e3.
3. Meyer, M.R., Maurer, H.M. (2010) Metabolism of designer drugs of
abuse: an updated review. Current Drug Metabolism, 11, 468482.
4. Marusich, J., Antonazzo, K., Wiley, J., Blough, B., Partilla, J., Baumann, M.
(2014) Pharmacology of novel synthetic stimulants structurally related
to the bath salts constituent 3,4-methylenedioxypyrovalerone
(MDPV). Neuropharmacology, http://dx.doi.org/10.1016/j/neuro
pharm.2014.02.016
5. Prosser, J.M., Nelson, L.S. (2011) The toxicology of bath salts: a review
of synthetic cathinones. Journal of Medical Toxicology, 8, 33 42.
6. Marinetti, L.J., Antonides, H.M. (2013) Analysis of synthetic cathinones
commonly found in bath salts in human performance and postmortem
toxicology: method development, drug distribution and interpretation
of results. Journal of Analytical Toxicology, 37, 135 146.
7. Moran, J., Seely, K. (2014) Bath saltsunderstanding a pervasive designer drug. Clinical Laboratory News, 40, 811.

Driving Case with a-PVP, Methylone and Ethylone 617