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Untargeted liquid chromatography-high resolution mass spectrometry (LC-HRMS) techniques have become indispensable tools for systematic toxicological analysis. They allow the research of an almost unlimited number of drugs within a single analytical cycle, but shared mass spectra libraries are still missing to identify newly marketed compounds, along with defined analytical procedures. This article describes the optimization, validation, and application of an untargeted screening method devoted to hair analysis using data-dependent analysis (DDA) and a shared HRMS database. This method used an ultra-high performance liquid chromatography coupled to a benchtop Orbitrap. Raw MS data were processed with Compound Discoverer software coupled to the mzCloud™ library. Optimizations were performed on blank hair spiked with 19 analytes having different physical and chemical properties. To validate the effectiveness of a shared spectra database, 20 compounds spectra were added and then retrospectively screened.Sensitivity and reliability were evaluated on 317 compounds of interest in toxicology. The method was then applied to 11 hair samples. The matrix effect range by ion suppression/enhancement was 40%-110%. The method allows the detection of 284 among the 317 screened compounds, including 72 new psychoactive substances (NPS).Lower limit of identification (LLOI) and lower limit of detection (LLOD) were 1 to 1000 pg/mg and 1 to 500 pg/mg, respectively. The method was successfully applied to 11 clinical cases and 144 compounds were identified including 24 NPS including AKB48-5F for the first time in hair. We developed and validated an LC-HRMS untargeted screening of 284 compounds and successfully applied it to 11 real hair samples.

Purpose To demonstrate the usefulness of hair testing to determine exposure pattern to fentanyls. Methods A 43-year-old male was found unconscious with respiratory depression 15 min after snorting 3 mg of a powder labeled as butyrylfentanyl. He was discharged from hospital within 2 days without blood or urine testing. Two locks of hair were sampled 1 month (M1 A: 0-2 cm (overdose time frame); B: 2-4 cm; C: 4-6 cm) and 1 year (Y1: A: 0-2 cm; B: 2-4 cm) later to monitor his exposure to drugs of abuse by liquid chromatography-tandem mass spectrometry after liquidliquid extraction. Results Hair analysis at M1 showed a repetitive exposure to 3-fluorofentanyl (A/B/C: 150/80/60 pg/mg) with higher concentration in segment A reflecting the overdose period. The non-detection of butyrylfentanyl was consistent with the analysis of the recovered powder identified as 3-fluorofentanyl. Furanylfentanyl (40/20/15 pg/mg) and fentanyl (37/25/3 pg/mg) were also detected in hair. The second hair analysis at Y1 showed the use of three new fentanyls, with probably repetitive exposures to methoxyacetylfentanyl (A/B: 500/600 pg/mg), and single or few exposures to carfentanil (2.5/3 pg/mg) and acetyl fentanyl (1/1 pg/mg). A decreasing exposure to 3-fluorofentanyl (25/80 pg/mg), and increasing consumption of furanylfentanyl (310/500 pg/mg) and fentanyl (620/760 pg/mg) were also observed despite methadone treatment initiation. The patient claimed not consuming three out of the six detected fentanyls. Conclusions We report single or repetitive exposure to several fentanyls using hair testing. To our knowledge, this is the first demonstration of 3-fluorofentanyl and methoxyacetylfentanyl in hair samples collected from an authentic abuser.Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

This study was to validate a LC–MS/MS method for the determination of 17 new synthetic opioids (NSOs) in hair including 3‐fluorofentanyl, 3‐methylfentanyl, acetylfentanyl, acetylnorfentanyl, alfentanyl, butyrylfentanyl, butyrylnorfentanyl, carfentanil, fentanyl, furanylfentanyl, furanylnorfentanyl, methoxyacetylfentanyl, norcarfentanil, norfentanyl, ocfentanil, sufentanil, and U‐47700, and to apply it to 137 authentic samples. Twenty milligrams of hair was decontaminated in dichloromethane and underwent liquid extraction. 10 μL of the reconstituted residue were injected onto the system. The separation was performed in 12 minutes in a gradient mode at a flow rate of 300 μL/min using a Hypersyl Gold PFP column (100 × 2.1 mm i.d., 1.9 μm) maintained at 30°C. Compounds were detected in positive ionization and MRM modes using a TSQ Endura mass spectrometer (ThermoFisher). The method was validated according to EMA guidelines. The LLOQ was in the range 1–50 pg/mg, and the calibration ranged from the LLOQ‐1000 pg/mg. Intra‐ and inter‐day accuracy (bias) and precision were < 15%. Extraction recoveries of parent drugs and metabolites were 74–120% and 7–62%, respectively. The matrix effect was in the range 59–126% (CVs ≤ 12.9%). Fentanyl was found in six cases at concentrations of < 1–1650 pg/mg (n = 14 segments). Five fentanyl analogs were quantified in two cases: 3‐fluorofentanyl (25–150 pg/mg, n = 5), furanylfentanyl (15–500 pg/mg, n = 5), methoxyacetylfentanyl (500–600 pg/mg, n = 2), acetylfentanyl (1 pg/mg, n = 2), carfentanyl (2.5–3 pg/mg, n = 2). This fully validated method allowed us to test for the first time 3‐fluorofentanyl and norcarfentanil in hair among 15 other NSOs, and brings new data regarding 3‐fluorofentanyl and methoxyacetylfentanyl hair concentrations.