Abstract

We present three fatal intoxications of methylone, a cathinone derivative. Blood was analyzed with a routine alkaline liquid–liquid extraction and analyzed by gas chromatography coupled with a mass spectrometer (GC–MS). Methylone was identified by a full scan mass spectral comparison to an analytical standard of methylone. For a definitive and conclusive confirmation and quantitation, methylone was also derivatized with heptafluorobutyric anhydride and analyzed by GC–MS. In all three fatalities, the deceased exhibited seizure-like activity and elevated body temperatures (103.9, 105.9 and 107°F) before death. Two of the three cases also exhibited metabolic acidosis. One of the three cases had prolonged treatment and hospitalization before death with symptoms similar to sympathomimetic toxicity, including metabolic acidosis, rhabdomyolysis, acute renal failure and disseminated intravascular coagulation. The laboratory results for this patient over the 24 h period of hospitalization were significant for increased lactate, liver transaminases, creatinine, myoglobin, creatine kinase and clotting times, and decreased pH, glucose and calcium. Peripheral blood methylone concentrations in the three fatal cases were 0.84, 3.3 and 0.56 mg/L. In conlusion, peripheral blood methylone concentrations in excess of 0.5 mg/L may result in death due to its toxic properties, which can include elevated body temperature and other sympathomimetic-like symptoms.

Introduction

Cathinone, an alkaloid structurally similar to amphetamine, was originally extracted from the fresh leaves of the Catha edulis or khat plant, which is native to east Africa and the Arabian Peninsula (1). Synthetic structural modifications of cathinone have led to a number of so-called designer cathinone derivatives that are commonly sold as “bath salts” on the internet, in smoke shops or in specialty head shops. These cathinone derivatives, namely, mephedrone, methedrone, methylone and 3,4-methylenedioxypyrovalerone (MDPV), have become increasingly available and abused in the United States. The number of alarming case reports from emergency rooms, poison centers and medical examiners involving severe reactions, including death, has prompted several states and the federal government to ban or schedule the bath salts. In 2011, the American Association of Poison Control Centers (AAPCC) reported 6,072 bath salt-related calls, compared to only 303 in 2010 (2).

As with amphetamines, cathinones act as central nervous system stimulants by increasing monoamine neurotransmitter concentrations. This action is a result of at least two distinct mechanisms: one involving the inhibition of monoamine neurotransmitter reuptake and the other involving drug-evoked release of monoamine neurotransmitters from intracellular storage vesicles (3). Similar to the amphetamines, individual cathinones may vary in their potencies on each of the three monoamine neurotransmitter pathways. Methylone effects are similar to methamphetamine, with the greatest potency on the inhibition of noradrenergic reuptake, followed by inhibition of serotonergic and dopaminergic reuptake (3, 4). Similar to methamphetamine, methylone is also a potent releaser of intracellular dopamine, serotonin and norepinephrine (5).

Mild clinical effects of the cathinones include tachycardia, palpitations, agitation, lack of appetite, increased alertness, anxiety and mydriasis. Severe symptoms include seizures, hyperthermia, hallucinations, nausea and vomiting, muscle spasms, rhabdomyolysis, renal failure, arrhythmia and death (1, 6, 7, 8, 9).

A handful of fatalities resulting from methedrone, mephedrone and MDPV toxicity have been reported; however, there is little information regarding methylone and its potential toxicity. In this case report, we present three fatalities involving methylone.

Case Histories

Case 1

At 5:30 p.m. on a Saturday evening, a 23-year-old male was walking in and out of traffic at a major intersection, banging on cars with fists, screaming profanities and acting erratically. He was yelling out rap song lyrics and speaking different languages. When approached by police, he refused to cooperate and became combative. He was taken down to the ground, subdued, handcuffed and transported to a local hospital emergency department. At the emergency room, he was combative, resisted removal from the police car and displayed unusual strength, requiring five individuals to help secure him to a hospital gurney.

Upon admission into the ER, he was restrained, combative and diaphoretic with a body temperature of 105.9°F. He was orally intubated for airway protection and sonorous respirations. Results from a computerized tomogram (CT) of the head and chest were unremarkable. He was bleeding from a tongue laceration (bite mark) that might have been caused from an acute seizure. He was placed under a cooling blanket and administered intravenous fluids containing antibiotics and pressors to treat possible septic shock and hypotension. Initial diagnoses were probable drug overdose, rhabdomyolysis, acute renal failure, acute respiratory failure, fever, confusional state and seizure. Approximately 3.5 h after admission, he went into cardiac arrest and, with cardiopulmonary resuscitation, was converted to sinus tachycardia. He went into cardiac arrest four more times over the next two hours. He developed disseminated intravascular coagulation (DIC), thrombocytopenia, anoxic encephalopathy and metabolic acidosis (without an osmolal gap). His final cardiac arrest occurred approximately 24 h after admission and he was pronounced dead. Clinical laboratory values over the 24 h course of the hospitalization are presented in Table I, and were remarkable for increased lactate, liver transaminases, creatinine, myoglobin, creatine kinase and clotting times, and decreased pH, glucose and calcium. At autopsy, no extrinsic disease was noted, other than a moderately hypertrophied left ventricle and obesity.

Table I

Clinical Laboratory Results for Case 1 during 24 Hours of Hospitalization*

Analysis 04/02/2011 1930 h 04/02/2011 2149 h 04/02/2011 2207 h 04/03/2011 0125 h 04/03/2011 0039 h 04/03/2011 0303h 04/03/2011 0827 h 04/03/2011 1010 h 04/03/2011 1414 h 04/03/2011 2008 h 
PO2 (75–110) mm Hg  286   289   275 185  
PCO2 (35–45) mm Hg  44   28   40 53  
pH (7.35–7.45)  7.31   7.32   7.23 7.13  
HCO3 (24–27) mEq/L  22   15   17 18  
Patient temp °F 105.7     98.6     
Na (135–148) mEq/L 141   141  141 150   149 
K (3.7–5.3) mEq/L 6.0   5.3  4.5 7.9  8.1 9.8 
Calcium (8.5–10.5) mg/dL 9.8   8.1  7.2 7.3   11.1 
Chloride (96–107) mEq/L 99   108  103 99   103 
Glucose (70–115) mg/dL 49   124  545 261   212 
Magnesium (1.6–2.6) mg/dL    3.5  3.4 4.4   3.9 
Phosphorus (2.7–4.5) mg/dL      9.1 16.0   20.5 
Lactic acid (0.5–2.2) mMol/L  4.5   8.9      
CO2 (21-31) mg/dL 19   18  20 19   
BUN (6–20) mg/dL 18   26  24 21   17 
Creatinine (0.5–1.2) mg/dL 2.7   4.2  4.0 4.6   5.0 
BUN/creatinine ratio 6.7   6.2  6.0 4.6   3.4 
Estimated GFR (mL/min) 29   18  19 16   14 
Osmolality (280–300)   313        
AST (5–40) IU/L 74   1,855  2,976 4,613   10,105 
ALT (5–40) IU/L 40   1,515  3,406 6,196   5,132 
Total bilirubin (0.0–1.0) mg/dL 0.9   1.4  0.7 1.3   0.8 
Alkaline phosphatase (39–117) U/L 87   62  33 53   104 
Total protein (6.1–7.9) g/dL 8.5   3.7  1.8 3.1   1.3 
Albumin (3.9–4.8) g/dL 4.7   1.7  <1.0 1.5   <1.0 
Anion gap 23   15  18 32   37 
Hemoglobin (14.1–18.1) gm/dL     13.7      
White blood cell count (4.6–10.2) K/UL 9.0     11.7 9.4  8.2 5.4 
Red blood cell count (4.69–6.13) M/UL 5.17     1.80 1.96  2.61 1.90 
Hemoglobin (14.1–18.1) g/dL 15.0     5.0 5.5  7.4 5.1 
Hematocrit (43.5–53.7) % 44.6     15.6 17.9  23.2 16.7 
Platelet count (142–424) K/UL 210     45 144  58 28 
Differential % granulocytes (39–77) % 70.2     68 59  75 42 PMN 17 BANDS 
Differential % lymphocytes (15–47) % 22.9     20 23  17 27 
Differential % monocytes ( 3–13) % 5.7     10  
CKMB (<5.3) ng/mL 17.3     174.8     
Myoglobin (<107) ng/mL >500  553,558        
Troponin (0.4) ng/mL 0.18     7.421     
CPK (<171)  2518    48,262     
Protime (9.0–12.5) s 11.3     83.1 22.7  >88.8  
APTT (21.0–34.0) s 21.5     >170.0 100.0  >170.0  
Analysis 04/02/2011 1930 h 04/02/2011 2149 h 04/02/2011 2207 h 04/03/2011 0125 h 04/03/2011 0039 h 04/03/2011 0303h 04/03/2011 0827 h 04/03/2011 1010 h 04/03/2011 1414 h 04/03/2011 2008 h 
PO2 (75–110) mm Hg  286   289   275 185  
PCO2 (35–45) mm Hg  44   28   40 53  
pH (7.35–7.45)  7.31   7.32   7.23 7.13  
HCO3 (24–27) mEq/L  22   15   17 18  
Patient temp °F 105.7     98.6     
Na (135–148) mEq/L 141   141  141 150   149 
K (3.7–5.3) mEq/L 6.0   5.3  4.5 7.9  8.1 9.8 
Calcium (8.5–10.5) mg/dL 9.8   8.1  7.2 7.3   11.1 
Chloride (96–107) mEq/L 99   108  103 99   103 
Glucose (70–115) mg/dL 49   124  545 261   212 
Magnesium (1.6–2.6) mg/dL    3.5  3.4 4.4   3.9 
Phosphorus (2.7–4.5) mg/dL      9.1 16.0   20.5 
Lactic acid (0.5–2.2) mMol/L  4.5   8.9      
CO2 (21-31) mg/dL 19   18  20 19   
BUN (6–20) mg/dL 18   26  24 21   17 
Creatinine (0.5–1.2) mg/dL 2.7   4.2  4.0 4.6   5.0 
BUN/creatinine ratio 6.7   6.2  6.0 4.6   3.4 
Estimated GFR (mL/min) 29   18  19 16   14 
Osmolality (280–300)   313        
AST (5–40) IU/L 74   1,855  2,976 4,613   10,105 
ALT (5–40) IU/L 40   1,515  3,406 6,196   5,132 
Total bilirubin (0.0–1.0) mg/dL 0.9   1.4  0.7 1.3   0.8 
Alkaline phosphatase (39–117) U/L 87   62  33 53   104 
Total protein (6.1–7.9) g/dL 8.5   3.7  1.8 3.1   1.3 
Albumin (3.9–4.8) g/dL 4.7   1.7  <1.0 1.5   <1.0 
Anion gap 23   15  18 32   37 
Hemoglobin (14.1–18.1) gm/dL     13.7      
White blood cell count (4.6–10.2) K/UL 9.0     11.7 9.4  8.2 5.4 
Red blood cell count (4.69–6.13) M/UL 5.17     1.80 1.96  2.61 1.90 
Hemoglobin (14.1–18.1) g/dL 15.0     5.0 5.5  7.4 5.1 
Hematocrit (43.5–53.7) % 44.6     15.6 17.9  23.2 16.7 
Platelet count (142–424) K/UL 210     45 144  58 28 
Differential % granulocytes (39–77) % 70.2     68 59  75 42 PMN 17 BANDS 
Differential % lymphocytes (15–47) % 22.9     20 23  17 27 
Differential % monocytes ( 3–13) % 5.7     10  
CKMB (<5.3) ng/mL 17.3     174.8     
Myoglobin (<107) ng/mL >500  553,558        
Troponin (0.4) ng/mL 0.18     7.421     
CPK (<171)  2518    48,262     
Protime (9.0–12.5) s 11.3     83.1 22.7  >88.8  
APTT (21.0–34.0) s 21.5     >170.0 100.0  >170.0  

*Normal ranges are expressed in parentheses.

Case 2

A 19-year-old female was reportedly attending a concert at a club in Kansas City, MO, with several friends. Around 1:00 a.m., she was witnessed to take a pill known as “Molly.” Shortly thereafter, she collapsed, and then got up and danced for several minutes. She then sat down, complaining of “not feeling right,” and was witnessed to seize twice by an off-duty paramedic at the club. Each seizure was approximately 20 seconds long. Additional EMS personnel were called in, and the decedent was transported to a local hospital. She was in asystole upon arrival after having been given two units of epinephrine, two units of atropine and one dose of Narcan en route. Resuscitation efforts were continued for eight minutes after arrival. With no change in her condition she was pronounced dead. No signs of injury were noted and no froth cone was observed by hospital staff. While in the ER, her axillary temperature was 103.9°F and the code sheet indicated that she exhibited rigor. No admission blood or CT/X-ray scans were obtained. At autopsy, no extrinsic disease was noted.

Case 3

A 23-year-old male went out with friends to an after-hours club. A witness reported that the victim took LSD. The witness believed that the victim was having a “bad trip and freaking out.” Another witness reported that the victim was acting “irrational and sweating.” The victim also told a witness that he was “rolling on ecstasy.” Due to the victim's erratic behavior, management requested that he be removed from the club and they facilitated his removal by securing him to a chair with plastic food wrap. They wrapped the plastic food wrap around his chest and arms and then his legs to secure him to a chair and then placed the victim (in the chair) in the back of a van. The ambient air temperature outside the van was 77°F that day. The victim was left in the van for approximately three to four hours until a person (who was not at the after-hours club) located the victim. The victim was still bound to the chair with plastic food wrap and convulsing. 911 was called, paramedics responded and noted that the victim was seizing with an initial blood pressure of 63/37 and a weak heart rate of 132. He was given lorazepam, etomidate, succinylcholine, fentanyl and midazolam for treatment and intubation. He was immediately transported to a hospital. Upon arrival in the ER, advanced cardiac life support (ACLS) was initiated. His core body temperature was noted to be 107°F and he was acidotic with a lactic acid of 19.6 mmol/L and a blood pH of 7.04. He was given cooled intravenous fluids and placed under a cooling blanket. ACLS was continued with a total of 10 units of epinephrine administered and he was pronounced dead within 45 minutes after admission into the ER. At autopsy, no extrinsic disease was noted.

Experimental

Analysis

Methylone was initially identified in all three cases while performing a routine alkaline drug screen by gas chromatography–mass spectrometry (GC–MS). An unidentified peak containing a prominent m/z 58 was identified between the retention times of cotinine and caffeine. This unidentified peak had a mass spectrum with a prominent base ion of 58 and low abundance ions of 91, 121 and 149, identical to the mass spectra for methylone published in the March 2011 issue of ToxTalk (10). Subsequently, analytical standards for the bath salts, including methylone, mephedrone, methedrone and MDPV, were purchased, spiked into blank blood, extracted and analyzed by GC–MS. The methylone standard had the exact same retention time as the unidentified peak and an identical mass spectrum.

Due to the lack of any case reports or published methods in the scientific literature on methylone and the nondescript mass spectrum of methylone, a method was developed to confirm and quantitate methylone using a heptafluorobutyryl (HFB) derivative to change the mass spectrum to a more unique spectrum and shift the retention time for a more definitive and conclusive identification of methylone. In each case, methylone was confirmed by full scan electron impact (EI) GC–MS mass spectral analysis of underivatized methylone, full scan EI–GC–MS mass spectral analysis of the HFB derivative of methylone and quantitated using the HFB derivative of methylone. Figure 1 shows the EI mass spectrum for underivatized methylone and Figure 2 shows the more unique mass spectrum of the HFB derivative of methylone. Figure 3 shows the mass spectrum for the HFB derivative of the methylone-d3 internal standard.

Figure 1.

GC–MS full mass spectrum of methylone (underivatized) with prominent base ion of 58, with less abundant ions of 91, 121 and 149.

Figure 1.

GC–MS full mass spectrum of methylone (underivatized) with prominent base ion of 58, with less abundant ions of 91, 121 and 149.

Figure 2.

GC–MS full mass spectrum of HFB derivative of methylone with base ion of 149 and other minor ions of 91, 121, 210 and 254.

Figure 2.

GC–MS full mass spectrum of HFB derivative of methylone with base ion of 149 and other minor ions of 91, 121, 210 and 254.

Figure 3.

GC–MS full mass spectrum of HFB derivative of methylone-d3 with base ion of 149 and other minor ions of 91, 121, 213 and 257.

Figure 3.

GC–MS full mass spectrum of HFB derivative of methylone-d3 with base ion of 149 and other minor ions of 91, 121, 213 and 257.

Reagents and materials

High-performance liquid chromatography (HPLC) grade toluene, hexane and ethyl acetate were supplied by Thermo Fisher Scientific. Heptafluorobutyric anhydride (HFBA) was supplied by Restek. Trisodium phosphate was supplied by Sigma–Aldrich Chemical Company. Analytical standards of methylone and methylone-d3 were supplied by Cerilliant. Enzyme-linked immunoassay (ELISA) kits were supplied by Immunalysis.

Standards, calibrators and control preparation

An analytical standard solution (1 mg/mL) of methylone was diluted to a working concentration (2 µg/mL) with methanol and stored at –10°C. Calibrators were prepared by spiking 1 mL blank blood to final concentrations of 0.05, 0.10, 0.25, 0.50, 0.75, 1.0, 1.5 and 2.0 mg/L methylone. The internal standard, methylone-d3, was spiked at a final concentration of 0.3 mg/L. Controls were prepared in 1 mL blank blood using a separate control working solution prepared from a separate batch of the same methylone lot number at concentrations of 0.25, 0.75 and 1.5 mg/L.

Sample extraction and derivatization for confirmation and quantitation

The method developed is a slight modification of the standard amphetamine procedure at the Hillsborough County Medical Examiner Department. It was developed to identify and quantitate methylone, mephedrone, methedrone and MDPV bath salts simultaneously; however, for the purposes of this publication, we present only the methylone results. One-milliliter volumes of specimens were extracted with 2 mL saturated trisodium phosphate buffer and 1 mL toluene. Samples were rotated slowly for 1 h, followed by centrifugation to achieve layer separation. The upper organic layer was transferred to conical bottom test tubes and evaporated under nitrogen at 30°C. Samples were reconstituted in 0.5 mL hexane and 50 µL HFBA was added to each sample. Samples were derivatized in a heat block at 70°C for 1 h. After derivatization, samples were removed from the heat block and evaporated under nitrogen at 30°C. Samples were reconstituted in 1.0 mL of saturated trisodium phosphate buffer and 200 µL of ethyl acetate was added to each sample. Samples were vortexed at high speed for 20 seconds, the layers were allowed to separate and a portion (approximately 100 µL) of the upper ethyl acetate layer was transferred to autosampler vials for analysis by GC–MS.

Instrumentation and chromatographic conditions

A Thermo Scientific ISQ GC–MS equipped with an AS300 autosampler was used for analysis. Chromatograph separation was performed on an RTx-5MS (Crossbond 5% diphenyl–95% dimethylpolysiloxane) 0.25 µm × 0.25 mm i.d. × 30 m column from Restek. The injection port was set at 260°C (injection volume 2 µL in splitless mode). The intial oven temperature was 100°C, with no hold time, ramped at 12°C/min to 200°C, then ramped at 30°C/min to reach a final temperature of 300°C, which was held for 4 min. Helium was used as carrier gas at a flow rate of 1.0 mL/min. The transfer line temperature was 285°C.

EI was used as the ionization mode with an ion source temperature of 300°C and operated in selected ion monitoring (SIM) mode. Ions monitored for the HFB derivatives were methylone-HFB (m/z254, 110) and methylone-d3-HFB (m/z257, 113). Ions used for quantification are underlined. The methylone-HFB and methylone-d3-HFB only have two unique ions each; therefore, all positive methylone cases were also confirmed in full scan (both underivatized and derivatized with HFB) without the addition of internal standard for definitive identification and confirmation.

Method validation studies

The method was validated to assess calibration model, limit of detection, limit of quantitation, accuracy, precision, carryover and specificity. The calibration model was established by preparing a set of seven spiked calibrators (final concentrations of 0.10, 0.25, 0.50, 0.75, 1.0, 1.5 and 2.0 mg/L) plus a zero calibrator (blank) in five separate runs over five separate days. The calibration model was determined to be linear (unweighted with origin ignored) over the range of 0.1 to 2 mg/L with average r2 = 0.998. The limits of detection and quantitation were estimated by the lowest acceptable non-zero calibrator within 20% of the target concentration. The limit of detection was 0.05 mg/L and the limit of quantitation was 0.10 mg/L. Accuracy and precision were assessed by preparing and analyzing the low, medium and high controls (spiked at final concentrations of 0.25, 0.75 and 1.5 mg/L) in triplicate over five different days. The overall bias did not exceed 5% and the within-run or between-run precision did not exceed 10%. Carryover was assessed by running a blank matrix sample immediately following the highest calibrator. There was no carryover at 2.0 mg/L.

Interference and specificity studies were conducted by analyzing 10 different sources of blank blood (including some bloods from blood bank, antemortem blood, postmortem peripheral blood, postmortem heart blood and postmortem cavity liquid) without the addition of internal standard. No endogenous blood components interfered with the analysis or contributed more than 10% of the ion signal of the lowest calibrators. Specificity studies were conducted by spiking blank blood with a number of other commonly detected drugs at a high concentration. Specificity studies indicated no interferences from 62 of the most frequently detected drugs in toxicology casework, including amphetamines, benzodiazepines, opiates, cocaine and metabolites, sedatives, hypnotics and antidepressants. When various tissues were analyzed, matrix matched blank tissues were spiked with controls to ensure that there were no obvious matrix effects on the quantitation of tissues using the calibration curve prepared in blood.

Results

In Case 1, screening was performed on the hospital admission samples. No volatiles were detected. Immunoassay was negative for 12 classes of drugs (acetaminophen, barbiturates, benzodiazepines, cannabinoids, carisoprodol/meprobamate, cocaine metabolite, fentanyl, methadone, methamphetamine/MDMA, opiates, oxycodone and salicylates). Analysis of an alkaline extract by GC–MS detected methylone, dextromethorphan, cotinine, caffeine and lidocaine. No other drugs were detected. The dextromethorphan concentration was <0.02 mg/L.

In Case 1, methylone quantitation was performed on multiple antemortem and postmortem specimens. In the antemortem blood specimens, methylone concentrations were 0.70, 0.66, 0.60, 0.61 and 0.62 mg/L at 0, 3, 7, 23 and 25 h after admission, respectively. Methylone analysis was also performed on postmortem specimens with resulting methylone concentrations of 0.84 mg/L in iliac blood, 1.0 mg/L in heart blood, 1.4 mg/L vitreous humor, 12 mg/L in gastric contents and 0.55 mg/L in urine. The medical examiner concluded that the cause of death was methylone intoxication and the manner of death was accidental.

Case 2 originated in the Kansas City Medical Examiner's Office. No volatiles were detected. Immunoassay results were negative for nine classes of drugs (cocaine metabolite, cannabinoids, opiates, benzodiazepines, phencyclidine, amphetamines, barbiturates, methadone and propoxyphene). Analysis of an alkaline extract by GC–MS detected the presence of methylone and lamotrigine. No other drugs were detected. The femoral blood concentration of lamotrigine was 2.5 mg/L. Femoral blood was submitted to Hillsborough County Medical Examiner's Office for the quantitation of methylone with a resulting femoral blood methylone concentration of 3.3 mg/L. The medical examiner concluded that the cause of death was acute methylone intoxication and the manner of death was accidental.

In Case 3, the peripheral blood ethanol concentration was 0.03 g/dL and the vitreous humor ethanol concentration was 0.01 g/dL. No other volatiles were detected. Immunoassay was negative for nine classes of drugs (acetaminophen, barbiturates, carisoprodol/meprobamate, cocaine metabolite, methadone, methamphetamine/MDMA, opiates, oxycodone and salicylates). Immunoassay was positive for three classes of drugs: benzodiazepines, cannabinoids and fentanyl. Analysis of an alkaline extract by GC–MS detected methylone and caffeine. The midazolam, lorazepam and fentanyl were all administered by the hospital with therapeutic blood concentrations (0.020, 0.029 and 0.0021 mg/L, respectively). Based on history, additional targeted analyses for LSD, GHB and cannabinoids were performed and all findings were negative.

Case 3 had the following methylone concentrations: 0.56 mg/L peripheral blood, 0.58 mg/L heart blood, 0.92 mg/L vitreous humor, 4.5 mg/L gastric contents, 0.88 mg/Kg liver and 230 mg/L urine. The medical examiner concluded that the cause of death was methylone intoxication and the manner of death was undetermined (due to the possible deleterious effects resulting from the restraint of the individual to a chair with plastic food wrap for several hours).

The results of quantitative analyses for methylone for the three cases are summarized along with reported body temperatures in Table II.

Table II

Body Temperatures and Methylone Concentrations in the Three Presented Case Fatalities

Case Body temperature (°F) Methylone concentrations (mg/L or mg/Kg)
 
  Antemortem blood Peripheral blood Heart blood Liver Vitreous humor Urine Gastric 
105.9 0.70 0.84 1.0  1.4 0.55 12* 
103.9  3.3      
107  0.56 0.58 0.88 0.92 230 4.5* 
Case Body temperature (°F) Methylone concentrations (mg/L or mg/Kg)
 
  Antemortem blood Peripheral blood Heart blood Liver Vitreous humor Urine Gastric 
105.9 0.70 0.84 1.0  1.4 0.55 12* 
103.9  3.3      
107  0.56 0.58 0.88 0.92 230 4.5* 

*Only a portion of total gastric contents were submitted for analysis so the methylone concentration in gastric contents has limited interpretive value.

Discussion

Methylone was readily detected in all three cases by a routine alkaline drug screen by GC–MS. However, because the drug is so new and little is known about it, a second definitive and conclusive confirmation of methylone was performed by derivatization with HFB to change the mass spectrum and shift the retention time. This resulted in two unique and distinct methods for the confirmation of methylone, a protocol that is recommended in forensic toxicology whenever possible or practical.

An attempt was made to set up the quantitation method that was similar to our amphetamine method with the HFB derivative. During the method development phase, methylone was added to the amphetamine calibrators in an attempt to simultaneously quantitate amphetamines and methylone using both methamphetamine-d5 and MDMA-d5 as internal standards. However, methylone did not exhibit acceptable linearity and had poor precision and accuracy. After some experimenting, it was discovered that using methylone-d3 internal standard greatly improved linearity, accuracy and precision. Despite increasing HFBA concentrations, it was also discovered that the methylone procedure worked best when analyzed separately from the amphetamines and derivatized 30 degrees higher than the amphetamines. It is possible that there was some type of competition for derivatization between methylone and amphetamines when analyzed simultaneously.

Case 1 provided an extremely rare complete clinical course of pathophysiological changes resulting from methylone toxicity, including elevated body temperature, acidosis and the sequelae of renal failure, liver failure and disseminated intravascular coagulation. Usually, this type of information is not available for postmortem cases. Case 1 also had the great potential to provide some insight regarding the half-life and elimination rate of methylone with the sequential hospital samples drawn over the entire 24 h time period. However, upon analysis of those samples, the blood concentration did not change significantly over time. This could either be due to a long half-life or, more probably, because the patient's kidneys and liver were no longer functioning well at that time (as demonstrated by clinical values in Table I). In addition, the patient was receiving various fluids, drugs and blood products via five intravenous and intra-arterial catheters in the intensive care unit, so some of the hospital blood samples may be subject to dilution with artificial fluids. Dilution might also explain why the postmortem heart and peripheral blood specimens are slightly higher than the antemortem specimens. It is also possible that higher postmortem methylone concentrations may be a result of postmortem redistribution. Additional cases and results will be needed to assess both the elimination rate of the drug and whether it is subject to postmortem redistribution.

Interestingly, all three of our cases had elevated body temperature. In review of the scientific literature reporting fatalities involving other bath salts, information of body temperature is not typically available because subjects were either found dead or hospital records were not available. However, one reported fatality involving methedrone had a reported body temperature of 107°F and similar clinical course of multiple organ failure over the 16 h period proceeding death (11). In a recent study, repeated administrations of methylone and mephedrone produced a dose-dependent hyperthermia in rats (12). Mechanistically, it would be interesting to know whether all bath salts consistently produce high body temperatures, and whether this is an unusual finding, or perhaps a prognostic indicator of bath salt toxicity.

In Case 2, it appears that a relatively short period of time occurred from the time of alleged drug ingestion to the reported seizures and death. Lamotrigine, which was also identified in this case, has been shown to inhibit the reuptake of dopamine, norepinephrine and serotonin (13). This raises the possibility that an additive or synergistic effect between methylone and lamotrigine may have contributed to the rapid death in this case. Case 2 also had the highest methylone concentration and the lowest body temperature. In the other two cases with higher body temperatures and lower blood methylone concentrations, the individuals survived for several hours following ingestion of the drug. The differences in blood concentrations and clinical course might arise from different mechanisms of methylone toxicity, one in which high doses of methylone results in an acute rapid death and another in which lower doses may cause a more subacute toxic manifestation that includes extremely elevated body temperatures followed by multiple organ failure.

At the time the first case was analyzed, there were no reported cases of methylone intoxications in the published literature. Recently, there was a case reporting seizures and hyponatremia resulting from ethcathinone and methylone poisoning (8). The victim developed rhabdomyolysis and DIC, but survived. No toxicological examinations were performed. More recently, a fatality resulting from methylone and butylone was reported (14). Similar to our cases, the victim was febrile, comatose, tachycardic and hypertensive upon arrival and despite maximal supportive care, developed multi-organ failure and expired. Toxicology examinations qualitatively identified methylone and butylone in urine and pill capsules.

Reports of fatalities involving other bath salts with quantitative toxicological examinations have been reported. Scotland reported four fatalities with mephedrone blood concentrations of 0.98, 2.24, 0.13 and 0.24 mg/L (15, 16). The Netherlands reported a fatal mephedrone intoxication with a blood concentration of 5.4 mg/L (17). Sweden reported two fatal methedrone intoxications with blood concentrations of 13 and 9 mg/kg (11). Two abstracts presented at the 2011 Society of Forensic Toxicologists/The International Association of Forensic Toxicologist Joint Annual Meeting reported two fatal MDPV intoxications with blood concentrations of 0.44 and 1.0 mg/L, respectively (18, 19). This information on other bath salt cases was invaluable for the interpretation of the methylone concentrations in our cases.

As with all postmortem toxicology interpretations, the conclusions that these three cases resulted from methylone toxicity were based on the lack of any significant anatomical findings, individual case history and blood concentrations similar to other fatalities involving mephedrone, methedrone and MDPV. The methylone concentrations in these cases should assist in the interpretation of blood concentrations in other methylone-related cases.

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