Diphenidine and its metabolites in blood and urine analyzed by MALDI-Q-TOF mass spectrometry

Abstract

New psychoactive substances that are different from synthetic cannabinoids or cathinone derivatives have appeared very recently on illicit drug markets. In 2014, three reports were published on diphenidine distribution in Japan. The first study characterized diphenidine and 1-benzylpiperidine in a powdered product called ‘‘fragrance powder’’ [1], while the second determined diphenidine and 5-fluoro-AB-PINACA in a dubious herbal product [2]. The third study dealt with a fatal case in which a victim smoked a herbal product containing diphenidine together with a herbal product containing AB-CHMINACA and 5-fluoroAMB [3]. The postmortem distributions of these substances determined by gas chromatography–mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS-MS) indicated that the main cause of death was diphenidine poisoning [3]. To date, the toxicity and metabolism of diphenidine have not been studied in any scientific context because it is a research chemical [4] with very little history of use in humans [5]. To tentatively identify unreported molecules, such as metabolites, their exact masses with sensitivities better than 0.001 Da are necessary [6]. Matrix-assisted laser desorption ionization (MALDI) can achieve a softer ionization than electrospray ionization under some conditions, and hence is suitable for the detection of intact molecules. Therefore, in the present work we examined diphenidine and its metabolites by MALDI quadrupole time-of-flight mass spectrometry (Q-TOF-MS) with a resolution better than 0.001 Da. Diphenidine [1-(1,2-diphenyl-ethyl)piperidine]-HCl, acetonitrile (ACN), and ethanol that were suitable for LC– MS, and other chemicals of analytical grade were obtained from Wako Pure Chemicals (Osaka, Japan). a-Cyano-4hydroxycinnamic acid (CHCA) was obtained from SigmaAldrich (St. Louis, MO, USA), and amitriptyline, to be used as internal standard (IS) for diphenidine quantitation, was obtained from Astellas Pharma (Tokyo, Japan). Pure water with a specific resistance of 18 MX cm was used (Millipore, Bedford, MA, USA). Blood samples from healthy volunteers under permission were used as blank samples, and those spiked with several amounts of diphenidine were used as positive samples. The postmortem blood and urine samples of a victim intoxicated mainly with diphenidine were obtained at autopsy performed in our laboratory [3]. Individual stock solutions of diphenidine and amitriptyline were prepared separately by dissolving the appropriate amount of each compound in ethanol at 1 mg/ ml. The solutions were stored at -20 C. Working calibration solutions and quality control solutions were prepared daily by diluting the stock solution of diphenidine with blank blood at 1–100 ng/ml. Amitriptyline at 50 ng/ ml in blood was used as IS for the determination. Diphenidine and its metabolites in a sample were extracted and detected together. One microliter of IS and 59 ll of water were added to 20 ll of blood or urine placed in a tube (Eppendorf AG, Hamburg, Germany) using a MICROMAN M10 pipette (Gilson S.A.S., Villiers le Bel, France), and the mixture was centrifuged at 10,000 g for 3 min. The supernatant (72 ll) was placed in a new tube, & Kayoko Minakata kminakat@hama-med.ac.jp