Analytical and Bioanalytical Chemistry

, Volume 410, Issue 20, pp 4967–4978 | Cite as

Rapid determination of designer benzodiazepines, benzodiazepines, and Z-hypnotics in whole blood using parallel artificial liquid membrane extraction and UHPLC-MS/MS

  • Linda Vårdal
  • Gladys Wong
  • Åse Marit Leere Øiestad
  • Stig Pedersen-Bjergaard
  • Astrid Gjelstad
  • Elisabeth Leere Øiestad
Research Paper


Benzodiazepines (BZD) and Z-hypnotics are frequently analyzed in forensic laboratories, and in 2012, the designer benzodiazepines (DBZD) emerged on the illegal drug scene. DBZD represent a particular challenge demanding new analytical methods. In this work, parallel artificial liquid membrane extraction (PALME) is used for sample preparation of DBZD, BZD, and Z-hypnotics in whole blood prior to UHPLC-MS/MS analysis. PALME of BZD, DBZD, and Z-hypnotics was performed from whole blood samples, and the analytes were extracted across a supported liquid membrane (SLM) and into an acceptor solution of dimethyl sulfoxide and 200 mM formic acid (75:25, v/v). The method was validated according to EMA guidelines. The method was linear throughout the calibration range (R2 > 0.99). Intra- and inter-day accuracy and precision, as well as matrix effects, were within the guideline limit of ± 15%. LOD and LLOQ ranged from 0.10 to 5.0 ng mL−1 and 3.2 to 160 ng mL−1, respectively. Extraction recoveries were reproducible and above 52%. The method was specific, and the analytes were stable in the PALME extracts for 4 and 10 days at 10 and − 20 °C. No carry-over was observed within the calibration range. PALME and UHPLC-MS/MS for the determination of DBZD, BZD, and Z-hypnotics in whole blood are a green and low-cost alternative that provides high sample throughput (96-well format), extensive sample clean-up, good sensitivity, and high reproducibility. The presented method is also the first method incorporating analysis of DBZD, BZD, and Z-hypnotics in whole blood in one efficient analysis.

Graphical abstract


Designer benzodiazepines Benzodiazepines Z-hypnotics Parallel artificial liquid membrane extraction LC-MS/MS Whole blood samples 



The Research Council of Norway is acknowledged for financial support through grant no. 231917. Gerrit Middelkoop, Elianne Seeberg, Marit Langødegaard, and Elin Eliassen are gratefully acknowledged for their help with the method comparison.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2018_1147_MOESM1_ESM.pdf (509 kb)
ESM 1 (PDF 141 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Linda Vårdal
    • 1
  • Gladys Wong
    • 1
  • Åse Marit Leere Øiestad
    • 2
  • Stig Pedersen-Bjergaard
    • 1
    • 3
  • Astrid Gjelstad
    • 1
  • Elisabeth Leere Øiestad
    • 1
    • 2
  1. 1.School of PharmacyUniversity of OsloOsloNorway
  2. 2.Department of Forensic SciencesOslo University HospitalOsloNorway
  3. 3.Faculty of Health and Medical Sciences, School of Pharmaceutical SciencesUniversity of CopenhagenCopenhagenDenmark

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