Xander van Wijk (Presenter)
The University of Chicago
Bio: Dr. Xander van Wijk received his Bachelor of Science in Molecular Life Sciences from Maastricht University, The Netherlands, and a Master of Science in Biomedical Sciences from Hasselt University, Belgium, with a specialization in Clinical and Molecular Sciences. He obtained his Ph.D. in Medical Sciences in the Department of Biochemistry at the Radboud University Nijmegen, The Netherlands. His research there was focused on the role of the polysaccharide heparan sulfate in angiogenesis. Afterwards he moved to San Diego for postdoctoral training at the University of California San Diego in the Program of Excellence in Glycosciences. There he investigated the role of extracellular matrix molecules in bacterial infection. In 2015 he started a Clinical Chemistry Fellowship at the University of California San Francisco, where he focused on clinical toxicology and cardiovascular biomarkers. In October 2017, Dr. van Wijk joined the faculty at The University of Chicago as an Assistant Professor, and Assistant Director of Clinical Chemistry Laboratories.
Authorship: Xander M. R. van Wijk (1,2), Cassandra Yun (1), Annie Arens (3,4), Derrick Lung (4,5), Alan H.B. Wu (1), and Kara L. Lynch (1)
(1) Laboratory Medicine, University of California, San Francisco and Zuckerberg San Francisco General; (2) Department of Pathology, The University of Chicago; (3) Emergency Medicine, University of California, San Francisco; (4) California Poison Control System, San Francisco Division; (5) Emergency Medicine, San Mateo Medical Center.
Benzodiazepines are widely used for treatment of anxiety and insomnia, however, this class of drugs is also commonly abused. Many different benzodiazepines and analogs have been produced that are not FDA-approved. We developed a liquid chromatography high-resolution mass spectrometry method for detection of 16 of these so-called ‘designer’ benzodiazepines in urine. The limit of detection for most of these compounds ranged from 5 to 50 ng/mL, and minor negative matrix effects were observed only in some instances. With the exception of ketazolam, all compounds showed significant reactivity with the ThermoFisher CEDIA® benzodiazepine immunoassay. Although we recently encountered three designer benzodiazepines in clinical toxicology cases (clonazolam, etizolam, and phenazepam), we did not detect any in 211 urine samples that were previously determined benzodiazepine-positive by immunoassay.
Benzodiazepines are widely used for treatment of anxiety and insomnia, however, this class of drugs is also commonly abused. Many different benzodiazepines and analogs have been produced that are not FDA-approved. These ‘designer’ benzodiazepines are often sold on the so-called ‘dark web’ of the internet. The objective of this study was to develop a liquid chromatography high-resolution mass spectrometry (LC-HRMS) method for a number of commonly abused designer benzodiazepines. We recently encountered two designer benzodiazepines (etizolam and phenazepam) in clinical toxicology cases in the San Francisco Bay Area [1, 2] and we present here a case of dual ingestion of clonazolam and etizolam. We also determined the cross-reactivity of designer benzodiazepines with the ThermoFisher CEDIA® benzodiazepine immunoassay and studied the incidence of designer benzodiazepines in urine samples that were previously found positive using this immunoassay.
Standards. 3-Hydroxy-phenazepam, adinazolam, bromazepam, clonazolam, delorazepam, deschloroetizolam, diclazepam, estazolam, etizolam, flubromazepam, flubromazolam, ketazolam, N-desalkyl-flurazepam, nimetazepam, phenazepam, and pyrazolam were purchased from Cayman Chemical. Diazepam-d5 was purchased from Cerilliant and was used as an internal standard.
Sample preparation and LC-HRMS conditions. Urine samples were diluted 1:4 using a mixture of 98% mobile phase A and 2% mobile phase B (2% B). HRMS data was acquired with an ABSciex TripleTOF®5600 system in positive ion mode, collecting full scan data with IDA triggered acquisition of product ion spectra. Chromatographic separations were performed on a Phenomenex Kinetex 3.5 μm C18 column (50 x 3.00 mm, 2.6 µm). Mobile phase A was 0.05% formic acid in 5 mM ammonium formate. Mobile phase B was 0.05% formic acid in 50% methanol 50% acetonitrile. The elution gradient was ramped linearly from 2% to 100% B over 10 minutes. Data analysis was done using PeakView® and MasterView® software (version 2.0, AB Sciex).
Method verification. The method was verified by evaluating the limit of detection (LOD) and matrix effects according to Thoren et al . The LOD was determined by spiking different concentrations of drug standard into drug-free urine (5, 10, 25, 50, 100, 250, and 500 ng/mL). Samples were injected in duplicate and a drug was called positive if a) it met our scoring criteria as outlined in Colby et al , and b) the signal-to-noise-ratio was >20:1. Matrix effects (MEs) were determined by spiking drug standards at 100 or 500 ng/mL into a commercial drug-free urine preparation and into urine of 2 healthy volunteers, and comparing this to drug standard spiked into water. Samples were injected in triplicate and MEs were calculated as follows: (mean signal intensity in urine – mean signal intensity in water) / mean signal intensity in water x 100 %.
Clinical toxicology case. Serum samples from a case of dual ingestion of clonazolam and etizolam were prepared by protein precipitation using acetonitrile, drying of the sample, and reconstitution in 2% B. Tablets containing clonazolam or etizolam were crushed, incubated overnight in methanol, and diluted with 1:1000 with 2% B. LC-HRMS conditions were as described above.
Immunoassay cross-reactivity. Drug standard was spiked into drug-free urine at 200 ng/mL. Samples were analyzed in triplicate using the ThermoFisher CEDIA® High Sensitivity Benzodiazepine Assay.
Incidence study. 211 urine samples that were previously determined positive on the ThermoFisher Benzodiazepine Assay at Zuckerberg San Francisco General were collected and analyzed using the LC-HRMS method for designer benzodiazepines.
Method verification. We have currently verified the method for the following compounds: 3-hydroxy-phenazepam (LOD: 50 ng/mL; ME: 2 – 29%), adinazolam (LOD: 25 ng/mL; ME -18 – 22%), bromazepam (LOD: 25 ng/mL; ME: 39 – 89%), clonazolam (LOD: 250 ng/mL; ME: -20 – 1%), delorazepam (LOD: 25 ng/mL; ME: 7 – 38%), deschloroetizolam (LOD: 5 ng/mL; ME: -26 – -2%), diclazepam (LOD: 5 ng/mL; -7 – 21%), estazolam (LOD: 10 ng/mL; ME: -7 – 22%), etizolam (LOD: 5 ng/mL; ME: 5 – 29%), flubromazepam (LOD: 50 ng/mL; ME: 18 – 49%), flubromazolam (LOD: 25 ng/mL; ME: 3 – 25%), ketazolam (LOD: 5 ng/mL; ME: -2 – 17%), N-desalkyl-flurazepam (LOD: 25 ng/mL; ME: 34 – 84%), nimetazepam (LOD: 10 ng/mL; ME: -10 – 20%), phenazepam (LOD: 25 ng/mL; ME: 11 – 56%), and pyrazolam (LOD: 50 ng/mL; ME: -36 – -17%).
Clinical toxicology case. A 34-year-old male who had ingested designer drug tablets presented with symptoms similar to a benzodiazepine overdose with significant sedation, but without any airway compromise. He was mildly hypotensive overnight with miosis. The tablets he had ingested were found to contain 1.1 mg clonazolam and 2.4 mg etizolam for the first and second type of tablet, respectively. Both clonazolam and etizolam were found in the patient’s serum.
Immunoassay cross-reactivity. With the exception of ketazolam, all compounds showed significant cross-reactivity with the ThermoFisher CEDIA® High Sensitivity Benzodiazepine Assay. At the cut-off concentration of 200 ng/mL, the reactivity was as follows: 3-hydroxy-phenazepam: 99%; adinazolam: 190%; bromazepam: 110%; clonazolam: 139%; delorazepam: 192%; deschloroetizolam: 121%; diclazepam: 177%; estazolam: 208%; etizolam: 102%; flubromazepam: 243%; flubromazolam: 236%, ketazolam: 8%; N-desalkyl-flurazepam: 244%; nimetazepam: 124%; phenazepam: 154%; and pyrazolam: 143%.
Incidence study. None of the 211 analyzed urine samples, which were previously determined positive on the ThermoFisher Benzodiazepine Assay, were found to contain designer benzodiazepines that were included in our method. Although one sample contained N-desalkyl-flurazepam, this was likely due to ingestion of flurazepam, as hydroxy-ethyl-flurazepam was also found.
Conclusions & Discussion
We developed an LC-HRMS method for 16 designer benzodiazepines. The LOD for these compounds ranged from 5 to 50 ng/mL, with the exception of clonazolam (LOD: 250 ng/mL). Mostly positive MEs were observed, except for pyrazolam, for which the ME ranged from -36 to -17%. All compounds with the exception of ketazolam showed significant reactivity with the ThermoFisher CEDIA® High Sensitivity Benzodiazepine Assay. Although we recently encountered three designer benzodiazepines (clonazolam, etizolam, and phenazepam) in clinical toxicology cases, we did not detect any in 211 urine samples that were previously determined benzodiazepine-positive by immunoassay. We are currently looking into detection of metabolites of designer benzodiazepines in these samples, as well as the possibility of hydrolysis to remove glucuronidation.
References & Acknowledgements:
 A.M. Arens, X.M. van Wijk, K.T. Vo, K.L. Lynch, A.H. Wu, C.G. Smollin, Adverse Effects From Counterfeit Alprazolam Tablets, JAMA Intern Med 176(10) (2016) 1554-1555.
 K.T. Vo, X.M. van Wijk, A.H. Wu, K.L. Lynch, R.Y. Ho, Synthetic agents off the darknet: a case of U-47700 and phenazepam abuse, Clin Toxicol (Phila) 55(1) (2017) 71-72.
 K.L. Thoren, J.M. Colby, S.B. Shugarts, A.H. Wu, K.L. Lynch, Comparison of Information-Dependent Acquisition on a Tandem Quadrupole TOF vs a Triple Quadrupole Linear Ion Trap Mass Spectrometer for Broad-Spectrum Drug Screening, Clin Chem 62(1) (2016) 170-8.
 J.M. Colby, K.L. Thoren, K.L. Lynch, Optimization and Validation of High-Resolution Mass Spectrometry Data Analysis Parameters, J Anal Toxicol 41(1) (2017) 1-5.
IP Royalty: no
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