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: [email protected]

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 officer’s 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 difficulties laboratories face in implementing analytical methods that can effectively and efficiently identify and quantify these compounds. Pure reference standards of these drugs must first 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 confirmation 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 influence (DUI) of drugs. Three synthetic cathinones, which are often referred to generically as bath salts, were detected in the subject’s blood. The compounds were a-pyrrolidinovalerophenone (a-PVP), ethylone and methylone. The subject’s driving behavior, performance on field sobriety tests and other clinical signs and symptoms are summarized.

Case report Arrest A 34-year-old male was stopped by a law enforcement officer 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 officer observed the vehicle cross the center line three times in the span of 1.5 miles before initiating a traffic stop. The driver was observed to appear disoriented and confused. He was reported to have slurred speech, bloodshot watery eyes and difficulty dividing his attention. A portable breath test was administered with the result of 0.000. The officer then conducted standardized field 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 fluctuated 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 difficulty performing the finger-to-nose test. During this test, an individual is instructed to touch the tip of his nose with the tip of his finger. The subject used the pad of his finger 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

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officer 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 individual’s 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: 5–8.5 mm) and 7.5 mm in direct light (normal: 2–4.5 mm). Overall reaction to light was noted to be slow. Subsequent to the eye examination, the DRE checked the individual’s 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 60–90 BPM, it is seen that there is a somewhat elevated pulse. The subject had an elevated systolic blood pressure of 150/82 mmHg (normal: 120–140/70–90). The subject’s 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 findings, the DRE observed raised taste buds and thick white saliva on the tongue. The subject’s 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 confirmed 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 influence of central nervous system (CNS) stimulants, specifically 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 film 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 unconfirmed spectral match of a-PVP was found utilizing an uncertified 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 flame ionization detection, with no drug detected (LOQ ¼ 10 mg/L). The subject had stated use of Depakotew, and this testing was performed due to this admission. In order to confirm the presence of a-PVP and any other compounds classified 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 confirming these compounds. Preparation of the sample at NMS Labs begins by transferring 500 mL of blanks, calibrators, controls and specimens to tubes. Twenty-five 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 10–15 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-flight 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 limit—10 ng/mL). Mitragynine and

7-hydroxymitragynine (Kratom) are additional compounds that can be confirmed by this analysis, but were not detected (reporting limit—10 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 identified 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 first 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 officer and the DRE are consistent with what one might expect from an individual under the influence 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 officer, and again during the DRE evaluation. It has been observed that when a-PVP has been administered to mice, there is a significant 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 specifically. 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 findings further the observations of officers involved and are consistent with the DRE’s opinion of an individual’s 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 officer 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 drowning—a 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, 468–482. 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 salts—understanding a pervasive designer drug. Clinical Laboratory News, 40, 8–11.

Driving Case with a-PVP, Methylone and Ethylone 617

Suspected impaired driving case involving α-pyrrolidinovalerophenone, methylone and ethylone.

This is the first reported case of α-pyrrolidinovalerophenone (α-PVP), methylone and ethylone in a suspected impaired driving case in the state of Was...
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