An ultra-sensitive UHPLC-QqQ-MS/MS method for determination of 54 benzodiazepines (pharmaceutical drugs, NPS and metabolites) and z-drugs in biological samples
Section snippets
Author contributions
Olga Wachełko: Conceptualization, Methodology, Writing – Original draft preparation; Preparation of illustrations; Paweł Szpot: Conceptualization, Supervision, Writing – Reviewing and Editing; Kaja Tusiewicz: Writing – Original draft preparation; Preparation of illustrations; Karolina Nowak: Preparation of method and authentic forensic samples; Validation; Analysis of data; Agnieszka Chłopaś-Konowałek: Interpretation of toxicological analysis results of authentic samples; Marcin Zawadzki:
Chemicals
Water (Chromasolv® LC–MS), methanol (Chromasolv® LC–MS), ethyl acetate, n-hexane, diethyl ether and formic acid were purchased from Sigma-Aldrich (Steinheim, Germany); ammonium formate from Sigma-Aldrich (Bangalore, India); ammonium carbonate from Fluka (Buchs, Switzerland); buffer solutions (pH 7.4, 8, 9, 10, 11, 12 ± 0.05) were purchased from TarChem (Tarnowskie Góry, Poland); buffer solution (pH 13 ± 0.05) was purchased from Chempur (Gliwice, Poland); adinazolam (1 mg/mL; methanol),
Method development
For the development of an analytical method allowing the determination of benzodiazepines, z-drugs and metabolites with the highest sensitivity, parameters such as sample preparation procedure, the volume of biological material, the selection of internal standards and injection volume were optimized. While choosing a suitable sample preparation technique, liquid-liquid extraction at different pH ranges and with the use of different organic solvents (with various degrees of polarity) were taken
Comparison of chromatographic methods in determination of benzodiazepines and z-drugs in biological samples
The most sensitive, selective and accurate methods applied for benzodiazepines and z-drugs determination in biological samples, are gas and liquid chromatographic techniques coupled with mass spectrometry. Nowadays, due to the wide variety of benzodiazepines which are available on both, pharmaceutical and illegal marketplaces, there is the increasing need for development of multi-drugs methods in order to determine large amounts of different chemical compounds. For this reason, in Table 4 and
Conclusions
The developed and fully validated ultra-sensitive UHPLC-QqQ-MS/MS method enabled determination of 54 benzodiazepines (pharmaceutical drugs, NPS and their metabolites) and 3 z-drugs with one metabolite. Acquired LOQ values which stand by pg/mL and reduced sample volume allow very sensitive determination of before-mentioned compounds, which is suitable and very desirable especially in forensic toxicology analysis, as well as in clinical toxicology which is evidenced by the positive results of
Novelty statement
Developed method, that comprise the biggest number of benzodiazepines to date, enables trace analysis of those compounds (pg/mL). Sample volume is reduced to only 100 μL, hence this method can be implemented in both clinical and forensic toxicology.
Ethical statement
All procedures performed in this study were in accordance with the ethical standards of the national committee and with the 1964 Declaration of Helsinki. Whole blood, bile, urine and vitreous humor samples were sent to our institute for toxicological analysis. The blank biological samples (postmortem urine and blood) used to develop the method were collected in accordance with Ethical Committee approval (No. 333/14). This article does not contain any studies with living human participants or
Declaration of competing interest
The authors declare no conflicts of interest (including financial and personal) that might appear to influence the work reported in this paper.
Acknowledgements
The authors thank the SHIM-POL A. M. Borzymowski Company for the opportunity of collecting MS/MS spectra for 59 substances with the use of the UHPLC–QTOF-MS-MS 9030 system.
The research was carried out under the National Centre for Research and Development project no. POIR.01.01.01-00-0175/18.
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