Metabolism of the benzodiazepines norflurazepam, flurazepam, fludiazepam and cinolazepam by human hepatocytes using high-resolution mass spectrometry and distinguishing their intake in authentic urine samples

2019 ◽  
Vol 38 (1) ◽  
pp. 79-94 ◽  
Author(s):  
Shimpei Watanabe ◽  
Svante Vikingsson ◽  
Anna Åstrand ◽  
Volker Auwärter ◽  
Henrik Gréen ◽  
...  
2019 ◽  
Vol 65 (7) ◽  
pp. 862-870 ◽  
Author(s):  
Jeffrey D Whitman ◽  
Kara L Lynch

Abstract BACKGROUND Untargeted data acquisition on high-resolution mass spectrometers (HRMSs) has been used in clinical toxicology for screening and identifying unknown compounds in patient samples. A common modality for untargeted HRMS data acquisition is information-dependent acquisition (IDA), which analyzes the most abundant small molecules within an acquisition cycle. This process can potentially lead to false negatives of clinically relevant compounds at low concentrations. Sequential window acquisition of all theoretical fragment ion spectra (SWATH) has emerged as a method of unbiased, untargeted HRMS data acquisition in which no spectral data are lost. SWATH has yet to be optimized and assessed for use in clinical toxicology. METHOD We developed a variable-window SWATH method (vSWATH) and compared it to IDA by limit of detection studies in drug-supplemented urine (81 compounds) and against a retrospective cohort of 50 clinical urine samples characterized by LC-MS/MS. RESULTS vSWATH had a lower limit of detection than IDA for 33 (41%) drugs and metabolites added into urine samples. Both IDA and vSWATH were equivalent in discovering compounds from clinical urine samples and confirmed 26 additional compounds not previously discovered by targeted LC-MS/MS. Lastly, the unbiased acquisition of spectra in vSWATH allowed for identification of 5 low-abundance compounds missed by IDA. CONCLUSIONS This vSWATH method for clinical toxicology demonstrated equivalent analytical sensitivity and specificity for untargeted drug screening and identification in urine samples. vSWATH provided the additional benefit of collecting all tandem mass spectrometry spectra in a sample, which could be useful in discovering low-abundance compounds not discovered by IDA.


Author(s):  
Sarah L Belsey ◽  
Robert J Flanagan

Abstract The advent of hundreds of new compounds aimed at the substance misuse market has posed new analytical challenges. A semi-quantitative liquid chromatography–high resolution mass spectrometry (LC–HRMS) method has been developed to detect exposure to two novel stimulants, mephedrone and ethylphenidate, and selected metabolites. Centrifuged urine (50 µL) was diluted with LC eluent containing internal standards (mephedrone-d3, methylphenidate-d9, and ritalinic acid-d10, all 0.02 mg/L) (450 µL). Intra- and inter-assay accuracy and precision were within ± 15% and < 6% respectively, for all analytes. The limit of detection was 0.01 mg/L all analytes. Urine samples from mephedrone and ethylphenidate users were analyzed using immunoassay (amphetamine-group CEDIA) and LC–HRMS. Ethylphenidate, mephedrone, and selected metabolites all had low cross-reactivity (<1%) with the immunoassay. The median (range) amphetamine-group CEDIA concentration in urine samples from mephedrone users (N = 11) was 0.30 (<0.041–3.04) mg/L, with only one sample giving a positive CEDIA result. The amphetamine-group CEDIA concentration in the urine sample from an ethylphenidate user was <0.041 mg/L. Improving the detection of novel compounds is of increasing importance to enable accurate diagnosis and treatment. Immunoassay methods used for drug screening may be inappropriate and lead to false negative results. Conversely, detection of these compounds is possible through use of LC–HRMS and can provide information on the metabolites present after exposure to these drugs.


2016 ◽  
Vol 408 (18) ◽  
pp. 4845-4856 ◽  
Author(s):  
Madeleine J. Swortwood ◽  
Kayla N. Ellefsen ◽  
Ariane Wohlfarth ◽  
Xingxing Diao ◽  
Marta Concheiro-Guisan ◽  
...  

2016 ◽  
Vol 62 (1) ◽  
pp. 157-169 ◽  
Author(s):  
Xingxing Diao ◽  
Ariane Wohlfarth ◽  
Shaokun Pang ◽  
Karl B Scheidweiler ◽  
Marilyn A Huestis

Abstract BACKGROUND Despite increasing prevalence of novel psychoactive substances, no human metabolism data are currently available, complicating laboratory documentation of intake in urine samples and assessment of the drugs' pharmacodynamic, pharmacokinetic, and toxicological properties. In 2014, THJ-018 and THJ-2201, synthetic cannabinoid indazole analogs of JWH-018 and AM-2201, were identified, with the National Forensic Laboratory Information System containing 220 THJ-2201 reports. Because of numerous adverse events, the Drug Enforcement Administration listed THJ-2201 as Schedule I in January 2015. METHODS We used high-resolution mass spectrometry (HR-MS) (TripleTOF 5600+) to identify optimal metabolite markers after incubating 10 μmol/L THJ-018 and THJ-2201 in human hepatocytes for 3 h. Data were acquired via full scan and information-dependent acquisition triggered product ion scans with mass defect filter. In silico metabolite predictions were performed with MetaSite and compared with metabolites identified in human hepatocytes. RESULTS Thirteen THJ-018 metabolites were detected, with the major metabolic pathways being hydroxylation on the N-pentyl chain and further oxidation or glucuronidation. For THJ-2201, 27 metabolites were observed, predominantly oxidative defluorination plus subsequent carboxylation or glucuronidation, and glucuronidation of hydroxylated metabolites. Dihydrodiol formation on the naphthalene moiety was observed for both compounds. MetaSite prediction matched well with THJ-018 hepatocyte metabolites but underestimated THJ-2201 oxidative defluorination. CONCLUSIONS With HR-MS for data acquisition and processing, we characterized THJ-018 and THJ-2201 metabolism in human hepatocytes and suggest appropriate markers for laboratories to identify THJ-018 and THJ-2201 intake and link observed adverse events to these new synthetic cannabinoids.


2016 ◽  
Vol 9 (5) ◽  
pp. 680-698 ◽  
Author(s):  
Ariane Wohlfarth ◽  
Markus Roman ◽  
Mikael Andersson ◽  
Fredrik C. Kugelberg ◽  
Xingxing Diao ◽  
...  

Sign in / Sign up

Export Citation Format

Share Document