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Journal of Analytical Toxicology, Vol. 34, April 2010 Case Report Multiple-Drug Toxicity Caused by the Coadministration of 4-Methylmethcathinone (Mephedrone) and Heroin* Amber J. Dickson†, Shawn P. Vorce, Barry Levine, and Marilyn R. Past Division of Forensic Toxicology, Armed Forces Medical Examiner System, Armed Forces Institute of Pathology, Rockville, Maryland 20850-3125 Abstract An accidental death caused by the combined use of a new designer drug, 4-methylmethcathinone (mephedrone), and heroin is reported. A 22-year-old Caucasian male was found unresponsive in his living quarters and was transported to the hospital where he died. During autopsy, needle marks were found along the decedent’s lower legs and ankles. Investigators discovered the decedent and his roommate had been using “Black Tar” heroin and mephedrone. Routine toxicological analysis detected morphine in the decedent’s blood at 0.06 mg/L. Additionally, 6-acetylmorphine, morphine, codeine, and doxylamine were detected in his urine. A designer drug screen, employing a basic liquid–liquid extraction followed by pentafluropropionic anhydride derivatization, was used to isolate mephedrone from both blood and urine specimens. The derivatized extracts were analyzed by gas chromatography– mass spectrometry (GC–MS) operating in full-scan mode. Quantitative analysis of mephedrone was performed by GC–MS operating in selective ion monitoring mode using methamphetamine-d14 as an internal standard. Mephedrone was confirmed in the decedent’s blood and urine at 0.50 and 198 mg/L, respectively. The physiological and pharmacological effects of mephedrone and any associated toxicity have not been reported. However, because of its structural similarities with methcathinone and the high concentration in the decedent’s blood, the overall contribution of mephedrone to the death could not be minimized. Therefore, the medical examiner reported the cause of death as multiple-drug toxicity and the manner of death as accidental. Introduction According to the 2007 National Survey on Drug Use and Health, approximately 153,000 Americans aged 12 or older were current heroin users (2). In vivo, heroin is rapidly hydrolyzed to a pharmacologically active metabolite, 6-acetylmorphine (6-AM), which is then hydrolyzed to morphine (1). Heroin is excreted in the urine primarily as morphine and morphine glucuronides. Other opium alkaloids, synthetic artifacts, and cutting agents, such as quinine and diltiazem, can be found in illicit heroin (1,3). A high risk of overdose is associated with heroin because the purity of illicit heroin can vary from 10% to more than 80% based on geographical location (1). Cyanosis, drowsiness, stupor, hypotension, and bradycardia are signs of heroin toxicity. Death from heroin toxicity is caused by respiratory depression and failure (4). Any other central nervous system depressant that is coadministered, such as alcohol or benzodiazepines, dramatically increases the risk of a fatal overdose (3,4). 4-Methylmethcathinone (mephedrone) is a synthetic stimulant and the 4-methyl aromatic analogue of methcathinone. Mephedrone is commonly referred to on the streets as 4-MMC, MMCAT, and Crab, reportedly because of its fishy smell (5,6). Mephedrone can be found for sale on the internet as a research chemical or plant food and is also marketed as a “legal high” in head shops in the U.K. (7). Synthesized as a cathinone analogue, the structural similarities between mephedrone and methcathinone are displayed in Figure 1. Published data on mephedrone’s chemical properties, pharmacology, and toxicity were not found. Most of the known information about its effects can be discovered through internet drug user forums. Heroin is a semisynthetic diacetyl derivative of morphine first synthesized in 1874 and later used in 1898 as a pharmaceutical preparation (1). Currently, heroin is not approved or available for medical purposes in the U.S. and is listed by the Drug Enforcement Administration (DEA) as a Schedule I drug. * Disclaimer: The opinion or assertions herein are those of the authors and do not necessarily reflect the view of the Department of the Army, Navy, or the Department of Defense. This work was supported in part by the American Registry of Pathology. † Author to whom correspondence should be addressed. E-mail: [email protected]. 162 Figure 1. Structure of 4-methylmethcathinone (mephedrone) and methcathinone. Reproduction (photocopying) of editorial content of this journal is prohibited without publisher’s permission. Journal of Analytical Toxicology, Vol. 34, April 2010 Based on user experiences, the stimulant and euphoriant effects of mephedrone are comparable to drugs such as cocaine, ecstasy, and amphetamine (5). The most common routes of administration for mephedrone are oral and insufflation, although there have been some reports of intravenous and suppository administration (5). After administration of mephedrone, the initial onset of stimulus and euphoria typically lasts 10–30 min, depending on dose and route of administration (5). According to users’ experiences, some individuals may consume their entire mephedrone supply continuously in order to maintain the initial effects of the drug (5). Side effects of mephedrone reported by users include skin rashes, numbness, headache, and minor amnesia (5). More serious side effects noted after large doses of mephedrone were skin discoloration, light-headedness, cramping, and aches (5). Because mephedrone is a research chemical, there have been no published studies on its human or animal toxicity, addiction or acute overdose potential, long-term effects, or allergic reactions. Structural similarities with methcathinone and user reported experiences would suggest that mephedrone shares many of the same euphoric and stimulant effects. Recently, there were two reported deaths in Sweden and Denmark related to possible mephedrone use. One of the individuals was reported to have taken mephedrone, and the other individual was found to be in possession of mephedrone prior to death (5). Mephedrone was consequently banned in both Sweden and Denmark, although the presence of mephedrone in the two deaths was not confirmed by toxicology results (5). Currently, mephedrone is also banned in Finland, Israel, and Norway, but it is still legal in other countries such as the U.K. and U.S. (5). According to multiple sources from the DEA, mephedrone had not been encountered in the U.S. until June 2009 and is considered a rare drug of abuse. However, in July 2009, a DEA Microgram Bulletin (8) contained a mephedrone submission from Oregon, but the presence of mephedrone was not confirmed because no authenticated standard was available. For this reason, mephedrone is currently unscheduled in the U.S.; however, it could possibly be controlled under the Federal Analogue Act of 1986 because of its chemical similarities with methcathinone. This case report describes an accidental death from multipledrug toxicity of mephedrone and heroin as well as the development of a screening and quantitative method for mephedrone detection and quantitation in biological specimens. Case History The decedent, a 22-year-old Caucasian male, was found unresponsive in his living quarters in the U.S. After emergency medical services arrived on the scene, they initiated CPR and transported the man to a nearby hospital. At the hospital, all life-saving measures were unsuccessful, and the man was pronounced dead. During the autopsy, multiple needle marks were found along the medial aspect of the decedent’s lower legs and ankles. There were no other signs of physical trauma on the decedent’s body. After an investigative search of the dece- dent’s living quarters, the following items were discovered: drug paraphernalia, syringes, suspected heroin, and other controlled substances including 2,5-dimethoxy-4-(n)propylthiophenethylamine (2C-T-7). The decedent’s roommate admitted to possessing the following drugs: “Black Tar” heroin, mephedrone, and 2C-T-7. He also confessed to providing the heroin and mephedrone to the decedent. Based on the roommate’s statement, both of them administered mephedrone doses by insufflation and intravenous and oral routes prior to injecting heroin in the early morning hours before the decedent was found unresponsive. According to the medical examiner report, the cause of death was multiple drug toxicity and the manner of death was accidental. The 22-year-old male roommate submitted a urine sample that was sent to the Navy Drug Screening Laboratory in Jacksonville, FL (NDSL JAX) for routine drugs-of-abuse testing. An aliquot of his urine sample was later submitted to the Division of Forensic Toxicology, Armed Forces Medical Examiner System (AFMES) for special testing of 2C-T-7 and mephedrone. Experimental Reagents and materials All solvents were high-performance liquid chromatography grade and purchased from Fisher Scientific (Pittsburgh, PA). Monobasic and dibasic sodium phosphate, sodium acetate trihydrate, mepivicaine, and ethylmorphine were purchased from Sigma Aldrich (St. Louis, MO). Mephedrone and mephedroned3 were purchased from Toronto Research Chemicals (North York, ON, Canada). Pentafluropropionic anhydride (PFPA) was purchased from Aldrich (Milwaukee, WI). Concentrated hydrochloric acid (HCl) was purchased from EMD Chemicals (Gibbstown, NJ). Concentrated potassium hydroxide (KOH) pellets were also purchased from Fisher Scientific. Methamphetamine-d14 and mescaline-d9 were purchased from Cerilliant (Round Rock, TX). Mixed-mode silica-based solid-phase extraction columns (ZCDAU020) were purchased from United Chemical Technologies (Bristol, PA). Standard, calibrators, and control preparation For the mephedrone extraction and quantitative procedure, a stock standard of mephedrone was prepared in methanol and stored at –15°C in an amber vial. Calibrator samples were prepared in 2.0 mL of certified drug-free blood by spiking mephedrone at concentrations of 0.05, 0.10, 0.20, 0.50, 1.00, and 2.00 mg/L. A blood control was prepared from the same stock standard source in certified drug-free blood at 0.50 mg/L. Calibrator and control samples were prepared prior to each extraction. Mephedrone-d3 and methamphetamine-d14 spiking solutions were prepared for use as the internal standards at a concentration of 0.01 mg/mL. Negative and positive blood controls were included and extracted with each batch. Instrumentation Samples were screened and quantitated using an Agilent Technologies (Palo Alto, CA) 6890 gas chromatograph (GC) 163 Journal of Analytical Toxicology, Vol. 34, April 2010 coupled to a 5975 mass spectrometer (MS). The separation columns were from J&W (20 m × 0.18-mm i.d. × 0.18 µm, Rancho Cordova, CA) with helium carrier gas maintained at a constant flow of 1.0 mL/min. The MS source and quadrupole temperatures were held at 230°C and 150°C, respectively. The transfer line was set to 280°C. Full-scan EI MS data was collected over a mass range of m/z 42–550 and a detection threshold of 150. Identification of targeted analytes was accomplished using selected ion monitoring (SIM). The SIM ions collected were m/z 204, 160, 119 for mephedrone and m/z 211, 163 for methamphetamine-d14; the quantitative ions are underlined. Basic drug screen extraction Blood (1 mL) and urine (2 mL) samples were prepared by the addition of 3 mL of 0.1 M phosphate buffer (pH 6.0). An internal standard solution containing 0.01 mg/mL mepivacaine and ethylmorphine was added to each sample at a concentration of 0.50 mg/L. Blood samples were sonicated for 15 min using an ultrasonic bath. All samples were centrifuged at 3000 rpm for 10 min prior to extraction. Extraction columns were conditioned successively with 3 mL of methanol, 3 mL deionized water, and 2 mL of 0.1 M phosphate buffer (pH 6.0). Following sample application, the columns were washed with 2 mL of deionized water, 2 mL of 20% acetonitrile in deionized water, and 2 mL of 0.1 M acetic acid. The columns were dried under vacuum for 3 min. The final wash steps included 2 mL of hexane followed by 3 mL of methanol. The columns were dried under vacuum for 10 min. The analytes were eluted with 3 mL of dichloromethane/isopropanol/ammonium hydroxide (78:20:2), and eluates were evaporated under nitrogen at 25°C. The eluates were reconstituted in 50 µL of acetonitrile and then transferred to glass autosampler vials. Three microliters of sample was injected using a pulsed split program of 40 psi for 0.8 min at a 10:1 split and an inlet temperature of 250°C. An initial oven temperature of 70°C was held for 1 min before ramping at 20°C/min to 300°C. The column was held for 5.5 min at the final temperature of 300°C for a total run time of 18 min. Designer drug screen extraction Three milliliters of sample was aliquoted, and 75 µL of the internal standard solution containing 0.01 mg/mL methamphetamine-d14 and mescaline-d9 was added to each sample for a final concentration of 0.25 mg/L. Four drops of concentrated KOH and 5 mL of chlorobutane were added to all samples. Next, the samples were capped and mixed for 20 min on a rotomixer. The samples were then centrifuged for 10 min at 3000 rpm. The top organic layer was transferred with a glass pipette to clean 10-mL conical test tubes. Prior to drying down under nitrogen at 40°C, 50 µL of 10% methanolic HCl was added to each sample. The eluates were reconstituted in 50 µL of ethyl acetate and 50 µL of PFPA. Each tube was capped, vortex mixed, and incubated at 70°C for 15 min. The samples were again evaporated to dryness under nitrogen at 40°C, reconstituted with 25 µL ethyl acetate, and then transferred to glass autosampler vials. Three microliters of sample were injected using a split pro- 164 gram of a 10:1 split and an inlet temperature of 225°C. An initial oven temperature of 100°C was held for 1.5 min before ramping at 10°C/min to 235°C and then 35°C/min to 300°C. The column was held for 1.5 min at the final temperature of 300°C for a total run time of 18.0 min. Mephedrone extraction Two milliliters of sample, calibrators, or control was pipetted into clean, labeled 16 × 100-mm test tubes. Then 100 µL of the internal standard solution containing 0.01 mg/mL methamphetamine-d14 was added to each sample for a final concentration of 0.50 mg/L. Four drops of concentrated KOH and 5 mL of chlorobutane were added to all samples. Next, the samples were capped and mixed for 20 min on a rotomixer. The samples were then centrifuged for 10 min at 3000 rpm. The top organic layer was transferred with a glass pipette to clean 10 mL conical test tubes. Prior to drying down under nitrogen at 25°C, 50 µL of 10% methanolic HCl was added to each sample. The eluates were reconstituted in 50 µL of ethyl acetate and 50 µL of PFPA. Each tube was capped, vortex mixed, and incubated at 70°C for 15 min. The samples were evaporated to dryness under nitrogen at 55°C, reconstituted with 50 µL ethyl acetate, and then transferred to glass autosampler vials. One microliter of sample was injected using a pulsed split program of 40 psi for 1.0 min at a 30:1 split and an inlet temperature of 225°C. An initial oven temperature of 100°C was held for 1 min before ramping at 20°C/min to 200°C and then 40°C/min to 300°C. The column was held for 0.5 min at the final temperature of 300°C for a total run time of 9.0 min. Method validation Selectivity. The selectivity of the assay was determined by analyzing three sources each of negative blood and negative urine for potential endogenous and extraneous interferences. Accuracy and precision. The accuracy of the method was determined by comparing the measured mean quantitated results with the theoretical spiked concentrations of mephedrone. The within-day accuracy was measured using five aliquots at four different concentrations in a single extraction. The between-day accuracy was measured at 4 different concentrations over 10 separate extractions. The measured values could not deviate more than 20% from the theoretical concentrations and were expressed as percent difference. The within-day precision was measured using five aliquots at four different concentrations in a single extraction. The between-day precision was measured over 10 extractions at 4 different concentrations. The precision of the method was expressed as the coefficient of variation (CV) and could not exceed 15%. Linearity. The minimal criterion for acceptability of a method’s linearity is to have a line of regression with a correlation coefficient (r2) of 0.99 or greater. The linearity of the method was determined with a 7-point calibration curve evaluated over 10 extractions. Each calibration batch contained a negative control and four positive controls spiked at four different concentrations. The calculated value of each calibration point and control could not exceed 20% of the theoretical spike. The upper limit of linearity (ULOL) was established by measuring two aliquots at six different concentrations with a Journal of Analytical Toxicology, Vol. 34, April 2010 seven-point calibration curve. The ULOL was defined as the highest analyte concentration where the measured value was within ± 20% of the theoretical concentration and the qualifier ratios were within ± 20% of the average ion ratios based on the calibrators. Limits of detection (LOD) and quantitation (LOQ). The LODs and the LOQs were established by measuring two aliquots at five different concentrations with a seven-point calibration curve. The LOD was defined as the lowest analyte concentration where all qualifier ion ratios were within ± 20% of the average ion ratios established from the calibrators. The LOQ was defined as the lowest analyte concentration where the measured value was within ± 20% of the theoretical concentration and all qualifier ion ratios were within ± 20% of the average ion ratios based on the calibrators. Recovery. The recovery was determined by comparing an unextracted mephedrone sample to an extracted mephedrone sample in blood in which the internal standard was added post-extraction. Recovery was determined at four different concentrations throughout the linear range. The recovery values were reported as the percent of drug recovered during the extraction procedure. Specificity. A comprehensive panel of commonly encountered alkaline compounds and a designer drug panel were analyzed to determine if any chromatographic or mass spectral interference would occur with this method. Chromatographic criteria. The retention time (tR) of the analytes was required to be within ± 2% of the tR of the calibrator. Qualifier ion ratios of the analytes needed to be within ± 20% of the average ion ratios established from the calibrators. A chromatographic peak should be Gaussian in shape with tailing factors less than 2.0. The peaks were required to have resolution from any adjacent peaks so that the valley between peaks is < 10% of the analyte’s peak height. Results ± 20% of the theoretical concentration). Additionally, the qualifier ion ratios of the LOD and LOQ values were within ± 20% of the average ion ratios determined from the calibrators. Recovery. The mephedrone recovery was calculated to be greater than 80%. Specificity. There were no interferences observed. Case samples A standard postmortem toxicology panel was performed on the decedent’s blood and urine, which included headspace GC volatile quantitation, an immunoassay urine drugs-of-abuse panel, and a full-scan GC–MS basic drug screen. No volatiles were detected from the GC headspace volatile quantitation including ethanol at cutoff of 20 mg/dL. The urine screened positive by immunoassay for 6-acetylmorphine (6-AM) and was confirmed by GC–MS SIM at 0.53 mg/L. No 6-AM was detected in the blood by GC–MS at an LOQ of 0.005 mg/L. Morphine was detected in the urine by immunoassay and confirmed in blood and urine by GC–MS SIM at 0.06 mg/L and 27.13 mg/L, respectively. Codeine was detected by GC–MS SIM in the urine at 1.86 mg/L; no codeine was detected in the blood by GC–MS at an LOQ of 0.05 mg/L. The urine opiate confirmation procedure involved an acid hydrolysis step; therefore, the previously mentioned urine morphine and codeine values represent total drug concentrations. A full-scan GC–MS basic drug screen in the urine detected mephedrone, doxylamine, morphine, and 6-AM (Figure 2). A GC–MS SIM basic drug quantitation confirmed the presence of doxylamine in the urine at 1.99 mg/L; no doxylamine was detected in the blood by GC–MS at an LOQ of 0.05 mg/L. A fullscan GC–MS designer drug screen performed on the decedent’s blood and urine specimens confirmed the presence of mephedrone in both specimens. However, the GC–MS designer drug screen did not detect 2C-T-7 in the blood or urine at an LOD of 0.025 mg/L. A quantitative GC–MS SIM method was developed for the mephedrone confirmation using methamphetamine-d14 as the internal standard with an LOD of 0.010 mg/L and a ULOL of 5.0 mg/L. Mephedrone was confirmed in Table I. Validation Data for Mephedrone in Blood Method validation The mephedrone validation results including accuracy, precision, linearity, LOD, LOQ, and recovery are summarized in Table I. Selectivity. There were no interferences observed in the negative blood and negative urine matrices analyzed. Accuracy and precision. Precision and accuracy met all acceptance criteria. The within-day CVs (n = 20) were less than 6.3% for mephedrone, and between-day CVs (n = 42) were less than 9.9% for mephedrone. Accuracy was determined for each aliquot analyzed (n = 62), and no samples exceeded 15.8% of their theoretical value for mephedrone. Linearity. Linearity was established from 0.025 to 5.000 mg/L for mephedrone, and the r2 value was 0.9976. LOD and LOQ. The LOD was determined to be 0.010 mg/L. The LOQ was established at 0.025 mg/L and defined as the lowest calibrator. The LOQ values met the criteria for acceptability in regards to precision (CV < 15%) and accuracy (within Mephedrone Linearity (n = 10) 0.025–5.000 mg/L r2 0.9976 LOD 0.010 mg/L LOQ 0.025 mg/L Recovery (n = 4) > 80 % Precision Within-day (n = 20) Between-day (n = 42) CV < 6.3% CV < 9.9% Accuracy* Within-day (n = 20) Between-day (n = 42) 2.7–15.8% 0.2–12.4% * Expressed as % difference from the theoretical value. 165 Journal of Analytical Toxicology, Vol. 34, April 2010 both the decedent’s blood and urine at 0.50 mg/L and 198 mg/L, respectively. A standard drug-of-abuse toxicology panel was performed on the roommate’s urine specimen at the NDSL JAX. His urine screened positive by immunoassay for 6-AM and was confirmed by GC–MS SIM at 0.007 mg/L. The urine screened positive by immunoassay for opiates, and both total codeine and morphine were confirmed by GC–MS SIM at 0.30 and 7.67 mg/L, respectively. Special testing for 2C-T-7 and mephedrone was performed at the Division of Forensic Toxicology, AFMES. A GC–MS designer drug screen detected the presence of mephedrone, but 2C-T-7 was not detected at an LOD of 0.025 mg/L. Mephedrone was confirmed and quantitated in the urine by GC–MS SIM at 8.10 mg/L. Discussion base peak ion (m/z 119) used for the mephedrone confirmation. Instead, methamphetamine-d14 was used as the internal standard for the mephedrone confirmation because of its similar chemical structure. The detection and confirmation of mephedrone in this case is consistent with the roommate’s statement that he had provided the mephedrone to the decedent just prior to his death. The roommate also stated the decedent had administered the mephedrone by multiple routes of administration (insufflation, oral, and intravenous). Because there have been no other reported mephedrone concentrations in blood or other biological specimens, it is difficult to correlate the quantitative values or describe the effects that the decedent experienced with his mephedrone blood concentration. Additionally, the effects and toxicity of mephedrone would be difficult to predict because of the coadministration of heroin. Prior to the decedent’s death, the roommate stated that he had also provided heroin to the decedent and that the decedent had administered the heroin intravenously. Heroin has a short half-life of about 2–6 min in blood because it is rapidly deacetylated to 6-AM by blood esterases (9). The half-life of 6-AM, which is 6–25 min in blood, is longer than that of heroin (9). 6-AM is further hydrolyzed to morphine, which has a half-life of 2–3 h (9). The presence of 6-AM in the urine demonstrates that the decedent administered heroin prior to his death. 6-AM was not detected in the blood because it had already metabolized to morphine. Morphine was detected in the blood at 0.06 In this case, our laboratory’s standard postmortem toxicology panel would not have been able to detect the presence of the two designer drugs, 2C-T-7 and mephedrone, unless a specific test request was made. A full-scan GC–MS basic drug screen of the urine detected an unidentified peak at 6.405 min with a base peak of m/z 58. A mass spectrum match could not be made for this peak when using searchable reference libraries. Because of the unidentified peak in the base screen and the possible use of 2C-T-7 and mephedrone, the toxicology testing was expanded to include a GC–MS designer drug screen. With the ability to detect 32 drugs, including 2C-T-7, our GC–MS designer drug screen was further expanded to include a reference standard of mephedrone. An unextracted mephedrone reference standard was analyzed, both underivatized and derivatized with PFPA, using the GC–MS designer drug screen. The mass spectrum of mephedrone and mephedrone-PFPA (Figure 3) were added to a custom in-house reference library. Once identifiable by GC–MS, both extraction and instrumentation methods were developed in order to detect and confirm the presence of mephedrone. Additionally, a deuterated mephedroned3 reference standard was purchased to be used as the internal standard. Unfortunately, the mephedrone-d3 MS fragmentation pattern was very similar to mephedrone-d0, sharing the same base peak ion (m/z 119). Furthermore, they could not be separated chromatographically. For this reason, the use of mephedrone-d3 as the internal standard Figure 2. Gas chromatogram and full-scan mass spectrum of mephedrone extracted from the decedent’s was not possible because it was conurine using a basic drug screen. tributing to the abundance of the qualifier 166 Journal of Analytical Toxicology, Vol. 34, April 2010 mg/L and was present in the urine at 27.13 mg/L. Because 6AM was detected in the urine, it is most likely that the morphine found in the blood and urine was from the metabolism of 6-AM. In cases involving victims of heroin overdoses, their morphine blood concentrations are indicative of recent drug use (9). From prior chronic use of heroin, overdose victims’ urine, bile, and tissue morphine concentrations can represent both past and recent drug exposure (9). There are two reasons that could explain the presence of codeine in the urine. First, the decedent could have also taken codeine in addition to the other drugs. Another explanation is that 6-acetylcodeine or codeine may have been present as an impurity in the heroin (9). Illicit heroin is made from raw opium that primarily contains morphine and codeine. During the synthesis, morphine is acetylated to produce heroin, and any codeine present is also acetylated to form 6-actetylcodeine (10–12). The 6-acetylcodeine may have been metabolized to codeine, or the codeine was actually present unchanged in the heroin and therefore detected in the urine. In this case, there was a significant difference between the drug concentrations detected in the decedent’s urine versus his blood. The difference in the urine and blood drug concentrations led to notably high urine/blood ratios observed. Based on the medical examiner’s report, the decedent and his roommate administered mephedrone and heroin in the “early morning” hours on May 21, 2009. An unknown amount of mephedrone doses were administered by various routes of administration (insufflation, intravenous, and oral). This was followed by an intravenously administered dose of an unknown amount of heroin. The decedent was pro- nounced dead later that same day at 4:47 p.m. Based on this information, there was a significant time lapse between the multiple drug administrations and the decedent’s time of death. Therefore, it is difficult to draw any significant conclusions about the urine/blood ratios because of the unknown amount of mephedrone and heroin doses in addition to the multiple routes of administration. The decedent’s roommate stated that he had self-administered the mephedrone by multiple routes of administration, including insufflation, oral, and intravenous, prior to administering heroin. It is difficult to predict the effects of mephedrone and heroin in this case because it is hard to correlate drug concentrations in urine with effects as well as the fact that both drugs were recreationally administered. The fact that one individual died while the other individual survived after self-administration of mephedrone and heroin illustrates the complexity of predicting adverse outcomes after abuse of multiple drugs. Because mephedrone has a structural similarity to amine compounds, it was of interest to know if mephedrone would cross-react with either amphetamine or ecstasy (MDMA) urine drug screening kits. The following urine drug screening assay kits were used: Roche Abuscreen Online® Amphetamine (Indianapolis, IN), Siemens Emit® II Plus Amphetamine (Newark, DE), and Microgenics DRI® Ecstasy (Fremont, CA). Negative urine specimens were spiked at the following mephedrone concentrations: 100, 200, and 250 mg/L. The mephedrone samples were analyzed on two different urine drug screening instruments: a Hitachi 912 and a Hitachi Modular P (Tokyo, Japan). Both the amphetamine and ecstasy kits were calibrated at 0.50 mg/L with d-amphetamine and MDMA, respectively. All three of the previously mentioned mephedrone concentrations screened negative when using both amphetamine kits and the ecstasy kit. Therefore, a case that is screened for amphetamines and MDMA using the three kits would not screen positive if it contained mephedrone at a concentration of 250 mg/L or less in urine. Conclusions Figure 3. Gas chromatogram and full-scan mass spectrum of the pentafluoropropionic anhydride (PFPA) derivative of mephedrone extracted from the decedent’s blood. Typical non-fatal investigative cases in our laboratory are tested for volatile analysis and abused drug testing but are not tested with a full-scan GC–MS basic drug screen or full-scan GC–MS designer drug screen. Commonly used immunoassay screening kits proved to have no cross-reactivity with mephedrone. In addition, none of the three reference mass spectral libraries used in our laboratory included mephedrone. Therefore, if an investigative case was submitted in which the individual was known to be using mephe- 167 Journal of Analytical Toxicology, Vol. 34, April 2010 drone without a specific request for mephedrone testing, a negative test result may be reported. This case illustrates the importance of investigators and medical examiners relaying pertinent information about their cases to the forensic toxicology laboratory. Otherwise, new drugs not before encountered may be left unidentified, and medical examiners may report a death determination without complete information. The medical examiner in this case reported that the cause of death was attributed to multiple-drug toxicity associated with mephedrone and heroin use. This is the first known reported death in the U.S. attributed to the use of mephedrone as well as the combined use of mephedrone and heroin. 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