Successful quantification of 4′-methyl-α-pyrrolidinohexanophenone (MPHP) in human urine using LC–TOF-MS in an autopsy case

Forensic Toxicol (2016) 34:398–402 DOI 10.1007/s11419-016-0307-z SHORT COMMUNICATION Successful quantification of 40 -methyl-apyrrolidinohexanophenone (MPHP) in human urine using LC–TOF-MS in an autopsy case Kaori Shintani-Ishida1 • Yasuhiro Kakiuchi1 • Hiroshi Ikegaya1 Received: 8 December 2015 / Accepted: 16 January 2016 / Published online: 4 February 2016 Ó Japanese Association of Forensic Toxicology and Springer Japan 2016 Abstract The toxicological detection of the new synthetic cathinone 40 -methyl-a-pyrrolidinohexanophenone (MPHP) in urine samples has been impossible, because much of MPHP is metabolized before its excretion into urine. In this study, we successfully quantified unmetabolized MPHP in urine of an autopsy case using a sensitive method by liquid chromatography–time-of-flight-mass spectrometry. The quantification method showed good linearity in the range of 1.00–100 ng/mL, and the limit of detection was 0.5 ng/mL in human urine. In the autopsy case, the concentrations of MPHP in urine, plasma, and liver tissue samples were determined to be 60.1, 32.9 ng/mL, and 63.1 ng/g, respectively. Keywords 40 -Methyl-a-pyrrolidinohexanophenone  MPHP  Synthetic cathinone  Liquid chromatography– time-of-flight-mass spectrometry  LC–TOF-MS  Human urine Introduction The new designer drug 40 -methyl-a-pyrrolidinohexanophenone (MPHP) is a synthetic cathinone classified as an a-pyrrolidionophenone derivative (see reviews [1, 2]). MPHP was first identified in seized products as a drug of abuse in Germany in 2000 [3]. In Japan, MPHP has been detected in many types of seized products including mixed & Kaori Shintani-Ishida 1 Department of Forensic Medicine, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, 465 Kajii-cho, Kamigyo-ku, Kyoto 602-8566, Japan 123 dried plants called ‘‘herbs,’’ powder-type products called ‘‘bath salts,’’ and liquid-type products called ‘‘liquid aroma’’ because it was first identified in distributed illegal products in 2013 [4]. MPHP is presumed to exhibit stimulant effects and serious toxicity like other synthetic cathinones and amphetamines [2, 5, 6]. There is only one report on acute poisoning with toxic liver damage and rhabdomyolysis after MPHP intake [7]. In this poisoning case, MPHP was found at a concentration of 100 ng/mL in the patient’s serum by gas chromatography–mass spectrometry (GC– MS), while it could not be detected in urine because of its metabolism [7], which is consistent with the findings in a study on MPHP metabolites in rat urine [8]. MPHP could not be found in rat urine 24 h after administration with either 1 mg/kg, which corresponds to the common dose of abusers, or 20 mg/kg by GC–MS with a limit of detection of 100 ng/mL [8]. Conversely, the main urinary MPHP metabolite 40 -carboxy-a-pyrrolidinohexanophenone (40 carboxy-PHP) could be detected in both human [7] and rat [8] urine after MPHP administration. Therefore, to date, the toxicological detection of MPHP in urine by GC–MS seems to be possible only via its metabolites, including 40 carboxy-PHP, but these compounds are not commercially available [9]. Recently, Minakata et al. [10] reported that a matrix-assisted laser desorption ionization-quadrupole time-of-flight-mass spectrometry enabled the sensitive quantification of MPHP with a range of 2–100 ng/mL, although the samples used consisted of blood from volunteers with MPHP added as a reference. In the present study, we are the first to identify and quantify MPHP in human postmortem urine by using liquid chromatography– time-of-flight-mass spectrometry (LC–TOF-MS) in an autopsy case. Forensic Toxicol (2016) 34:398–402 Case history A 52-year-old man started to thrash about suddenly while sleeping and had a general convulsion. His wife and two children held down his arms and legs for approximately 20 min until his convulsion calmed down. At this point, his family assumed that the patient had fallen asleep again. They suspected abuse of designer drugs as a cause of his convulsion because he had been suspected of possession of illegal designer drugs and was questioned by the police 4 months earlier. The family then searched the living room for drugs and found eight packages consisting of one black package labeled ‘‘Zombie,’’ three clear packages ‘‘Bolt 1G,’’ ‘‘Aladdin pre 1G,’’ and ‘‘Spica 0.2’’ written on them, and four unlabeled clear packages. His wife returned to the bedroom and found him dead. An autopsy was performed at our department 1.5 days later. He had no notable medical history, but his daughter had seen him thrashing about approximately 1 month earlier. Alcohol was not detected in either blood or urine samples by GC. Testing of urine samples by a Triage DOA kit (Sysmex, Kobe, Japan) showed negative results. Screening of a forensic toxicology library (MassLynx 4.1 TOF Toxicology Database 1; Waters, Milford, MA, USA) using a LC–TOF-MS system (ACQUITY UPLC-XevoÒ G2-S QTof; Waters) detected nothing in the blood and urine samples. Materials and methods Reagents and materials MPHP and 40 -methoxy-a-pyrrolidinopropiophenone (MeOPPP) were purchased from Cayman Chemical Co. (Ann Arbor, MI, USA). Other common chemicals were of analytical grade and purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan). Blank blood and urine samples were collected from healthy volunteers with no history of drug intake after informed consent was obtained. The blank samples were screened for common drugs of abuse, alcohol, and MPHP and all were confirmed to be negative. Sampling and sample preparation Heart blood, urine, and liver tissue samples were collected at autopsy. The blood samples were centrifuged at 16009g for 10 min immediately after autopsy to prepare plasma. The plasma, urine and liver tissue samples were stored at -20 °C until use. 399 Liver tissue specimens were homogenized with 4 times (v/w) the amount of ultra-pure water using a bead beatertype shaking machine (TissueLyser II; QIAGEN, Hilden, Germany), and then the homogenate was diluted two-fold with ultra-pure water. Forty-five microliter samples of plasma, urine, or diluted liver tissue homogenate were deproteinized with 100 lL of acetone, and followed by the addition of 5 ll of 1.0 lg/mL MeOPPP as an internal standard. After centrifugation at 10,0009g for 5 min, the supernatant was used for LC–TOF-MS analyses. LC–TOF-MS conditions LC–TOF-MS conditions were exactly the same as described in our previous report [11]. Briefly, LC–TOF-MS was conducted on a Waters ACQUITY UPLC system with a quadrupole time-of-flight MS system (XevoÒ G2-S QTof; Waters). LC separation was performed with a Waters ACQUITY UPLC HSS C18 column (2.1 9 150 mm, 1.8 mm i.d., 1.8 lm particle size) with an aqueous solution of 5 mM ammonium formate adjusted to pH 3.0 using formic acid (solvent A) and an acetonitrile solution containing 0.1 % (v/v) formic acid (solvent B) for the mobile phases. The gradient elution mode was employed using the (...truncated)