Contents lists available at ScienceDirect Clinical Biochemistry journal homepage: www.elsevier.com/locate/clinbiochem Short Communication Rapid identification of cyclopropyl fentanyl/crotonyl fentanyl in clinical urine specimens: A case study of clinical laboratory collaboration in Canada Jan Palaty a, ⁎ , Danijela Konforte b , Theano Karakosta b , Ernest Wong c , Cristiana Stefan c a LifeLabs Medical Laboratories, Burnaby, British Columbia, Canada b LifeLabs Medical Laboratories, Toronto, Ontario, Canada c Centre for Addiction and Mental Health, Toronto, Ontario, Canada 1. Introduction The current worldwide opioid crisis continues to challenge both law enforcement laboratories evaluating seized drugs and clinical labora- tories monitoring use by patients. In particular, both facilities face the problem of identifying new street drugs as they are introduced to the illicit market such as the fentanyl (FEN) analogues that have been in- creasingly detected in overdoses and fatalities [1–6]. The controlled substance regulation processes drive the rapid changes of drugs on the street market, while analytical constraints related to technology and/or availability of certified standards delay the process of street drug identification. In this communication, we describe a collaborative strategy combining powerful and innovative mass spectrometry algo- rithms that led to the identification of an unexpected pair of fentanyl analogue isomers, cyclopropyl fentanyl (Cp-FEN)/crotonyl fentanyl and their metabolites, in a large number of urine samples from substance use disorder patients in two Canadian provinces. 1.1. Materials For LC/MS/MS, all solvents were from EMD Millipore (Billerica, MA, USA), Formic acid from Sigma-Aldrich (Oakville, ON, Canada) and stock solutions of fentanyl, fentanyl-D 5 , norfentanyl and norfentanyl-D 5 were from Cerilliant (Round Rock, TX, USA). Abalone glucuronidase was from KURA Biotec (Los Angeles, CA, USA): the BG100 product was used in Ontario (ON) and the initial British Columbia (BC) work, while KURA Turbo was used in the subsequent BC studies. Fentanyl urine screening was by EMIT (Enzyme Multiplied Immunoassay Technique) immunoassay from Thermo Scientific (Waltham, MA, USA) at a cut-off of 1 ng/mL. For high-resolution LC/MS/MS, methanol was from VWR International (Mississauga, ON, Canada), ammonium formate from Sigma-Aldrich and the internal standards morphine-D 3 and methadone- D 9 from Cerilliant. 1.2. Patient samples Patient samples were selected from those submitted to LifeLabs Medical Laboratories in BC or ON for routine urine fentanyl testing. Most samples originated in community-based substance use disorder clinics. 1.3. LC/MS/MS: sample preparation and instrument parameters The initial precursor ion scan studies were done by the BC lab. Sample preparation consisted of mixing 125 μL urine with 125 μL KURA BG100 glucuronidase (pH 4.8 0.1 M acetate buffer), hydrolysis (60 °C/ 15 min), salt-assisted liquid-liquid extraction (700 μL acetonitrile plus 100 μL brine), evaporation and reconstitution (200 μL 0.1% aqueous formic acid). Sample (25 μL) was loaded on a 2.1 × 50 mm Kinetex Biphenyl column (Phenomenex, Torrance, CA, USA) interfaced to an Agilent model 6410 tandem mass spectrometer (Santa Clara, CA, USA). Mobile phases consisted of 0.2% formic acid in water (solvent A) and 0.2% formic acid in methanol (solvent B). MS detection of Cp-FEN in- itially used a precursor ion scan of m/z 200–400 for fragments m/z 188 and m/z 84 using same source and collision cell conditions as fentanyl and norfentanyl. For multiple reaction monitoring (MRM), the instru- ment was set to transitions of m/z 337 > 188 (quantifier) and 337 > 105 (qualifier) for fentanyl, 342 > 188 for fentanyl-D 5 , 233 > 84 (quantifier) and 233 > 55 (qualifier) for norfentanyl, and 238 > 84 for norfentanyl-D 5 . In the subsequent BC studies with Cp- FEN, the procedure was modified as follows in order to enhance throughput via automation. Sample (200 μL) was heated with 400 μL pH 6.8 0.15 M phosphate buffer containing internal standards and KURA Turbo glucuronides for 30 min at 50 °C, following which 200 μL was applied to a 1 mL CEREX HP SCX mixed-mode SPE column (Tecan SP, Baldwin Park, CA, USA), which was then washed with water (700 μL), 0.1 M hydrochloric acid (300 μL) and water (500 μL) before drying and elution with dichloromethane/isopropanol/ammonium hy- droxide (70:28:2, v/v). Following reconstitution with water (200 μL), 20 μL was injected to the same column type as above with a gradient profile as follows: 5% B for 0.5 min, 5% to 25% B for 1.5 min, 25% B to 80% B for 3 min, 95% B for 0.5 min, 95% B to 5% B for 0.5 min. The same Agilent 6410 mass spectrometer was used with MRM settings as before, but with the addition of transitions of 349 > 188 (quantifier) https://doi.org/10.1016/j.clinbiochem.2018.01.013 Received 5 December 2017; Received in revised form 5 January 2018; Accepted 20 January 2018 ⁎ Corresponding author. E-mail address: jan.palaty@lifelabs.com (J. Palaty). Clinical Biochemistry xxx (xxxx) xxx–xxx 0009-9120/ Crown Copyright © 2018 Published by Elsevier Inc. on behalf of The Canadian Society of Clinical Chemists. All rights reserved. Please cite this article as: Palaty, J., Clinical Biochemistry (2018), https://doi.org/10.1016/j.clinbiochem.2018.01.013