Detection and semi-quantitative determination of designer benzodiazepines in serum using LC-MSn

Significance of the Topic

Designer benzodiazepines have rapidly emerged within the group of new psychoactive substances, presenting forensic and clinical toxicology with challenges in detection and quantitation. Their structural diversity—including precursors, active metabolites and hybrid derivatives—demands comprehensive screening methods capable of unambiguous identification at low concentrations in biological matrices. The integration of semi-quantitative workflows further enhances the interpretive power of toxicological analyses, guiding subsequent confirmatory testing and supporting casework decisions.

Objectives and Study Overview

This study aimed to extend a routine screening library (Toxtyper™) with spectra of 58 benzodiazepines (including 21 novel designer compounds and three z-drugs) and to establish a streamlined semi-quantitative protocol. Key objectives included the evaluation of limits of detection (LOD), matrix effects, linear dynamic range and the feasibility of single-point calibrations for rapid concentration estimates in serum.

Methodology and Used Instrumentation

• Sample Preparation: 1 mL human serum spiked with internal standards (D5-diazepam, D4-haloperidol, D3-doxepin at 50 ng/mL each). Alkaline liquid–liquid extraction with 1-chlorobutane and borate buffer (pH 9), evaporation under N₂ (40 °C), reconstitution in 25 µL 50:50 eluent A / B.
• LC Conditions: Dionex UltiMate 3000; Acclaim® RSLC 120 C18 column (2.1×100 mm, 2.2 µm); mobile phases A (water + 2 mM ammonium formate, 0.1% formic acid, 1% ACN) and B (ACN + 2 mM ammonium formate, 0.1% formic acid, 1% water); 500 µL/min; 2 µL injection; gradient elution (1%–95% B over 11 min, re-equilibration to 1% B).
• MS Conditions: Bruker amaZon speed™ ion trap; positive-ESI; full-scan UltraScan (70–600 Da); data-dependent AutoMSn (n=3) without polarity switching; scheduled precursor list disabled.
• Library Building: Direct infusion of research chemical standards; selection of highest-quality MS2/MS3 spectra; acceptance criteria: precursor mass Δ ≤ ±0.25 amu, MS2 base peak ≥10⁵ counts, MS3 base peak ≥10⁴ counts; metadata annotation (empirical formula, structure).
• Validation Experiments: LOD determined by automated positive identification in six replicates at descending concentrations. Matrix effects (ME) and recovery (RE) assessed at low (25–50 ng/mL) and high (250 ng/mL) levels in five volunteer sera. Linearity evaluated across 1–500 ng/mL (n=8) using peak area of [M+H]+ without IS normalization.

Main Results and Discussion

• Library Extension: 58 substances covering commonly prescribed, precursor and designer benzodiazepines, plus z-drugs.
• LOD: Majority at 5 ng/mL; nine compounds between 10–50 ng/mL; nifoxipam undetectable at trace levels due to instability.
• Matrix Effects: ME ranged from 53% to 244% (SD 1.9–33.6); recovery generally satisfactory except nitrazepam degradation indicated by ME 110% and RE 0%.
• Linearity: Most analytes linear from 5–500 ng/mL (R² >0.99 for 20 compounds; 38 compounds after single outlier removal).
• Semi-Quantitative Screening: Single-point calibrator at 50 ng/mL; IS-corrected area ratios automatically processed; results outside dynamic range flagged as < calLow or > calHigh; reproducibility within ±10–25% at mid-range.

Benefits and Practical Applications of the Method

• Automated identification combined with semi-quantitative data accelerates toxicological evaluation and prioritizes samples for confirmatory testing.
• Sub-therapeutic and low therapeutic concentration detection supports clinical and forensic investigations.
• Library flexibility allows rapid inclusion of emerging designer benzodiazepines according to user needs.

Future Trends and Applications

The continuous influx of novel benzodiazepine analogues will necessitate dynamic library updates and enhanced spectral deconvolution algorithms. Integration of high-resolution mass spectrometry and machine-learning–based spectral matching may further improve identification confidence. Expansion into other biological matrices and development of fully quantitative multi-analyte assays will broaden the method’s utility in clinical toxicology, doping control and environmental monitoring.

Conclusion

The presented workflow enables robust screening and semi-quantitative assessment of 58 benzodiazepines in human serum with detection limits suitable for forensic and clinical applications. By leveraging existing full-scan screening data, it provides rapid concentration estimates to guide analytical strategies, while allowing straightforward extension to accommodate new designer compounds.

References

• Demme C, Böttcher S, Meyer MR, Matuszewski BK. Poster presentation, TIAFT Meeting; 2005.
• Meyer MR, Kempf J, Peter R, Wilde M. Poster presentation (TP29), 61st ASMS Conference; 2013.
• Matuszewski BK, Constanzer ML, Chavez-Eng CM. Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC–MS/MS. Anal Chem. 2003;75(13):3019–3030.
• Meyer MR, Maurer HH. Identification and structural elucidation of phase I and II metabolites of new designer benzodiazepines in vitro and in vivo using high-resolution mass spectrometry. Anal Bioanal Chem. 2016;408(14):3571–3591.