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Development and validation of fast UHPLC-MS/MS screening method for 87 NPS and 32 other drugs of abuse in hair and nails: application to real cases


Interest on keratinized matrix analysis for clinical and forensic purposes has been recently grown due to the wide temporary detection window for psychotropic and toxic substances entrapped after repeated consumption. The aim of this study was the development and full validation of an UHPLC-MS/MS screening method to quantify 119 molecules among most abused classic drugs and new psychoactive substances in hair and in nails, to assess the polyconsumption. Twenty-five milligrams of hair or nail samples, added with the internal standard mixture, were cut and incubated with 500 μL M3® buffer reagent at controlled temperature. After cooling, 1 μL supernatant was injected in the chromatographic system equipped with an Oasis HLB column. After the 10 min chromatographic separation through a gradient mobile phase (aqueous ammonium formate, phase A; acetonitrile, phase B), the target compounds were detected in multiple reaction monitoring mode. The method was linear (r2 always better than 0.99) in a calibration range of LOQ 20000 pg compound for milligram hair and of LOQ 1000 pg compound per milligram nail. Process efficiency of analytes under investigation was always better than 65% and no significant ion suppression due to matrix effect was observed. Intra-assay and inter-assay precision and accuracy were always better than 15%. The applicability and trueness of the method were examined by analysing real samples of hair and nail from users of psychoactive drugs in recreational contexts. Both classic drugs and new psychoactive substances could be determined as result of single or repeated use and accumulation in keratin matrices.

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The authors would like to thank Simonetta di Carlo, Laura Martucci for the technical assistance and Soraya Martín (RN) for her valuable assistance throughout the clinical study. Oronzo Persano is acknowledged for manuscript editing and uploading.


The investigation was partially carried out with funding from Presidency of the Ministers Council, Department of Antidrug Policy and from Department of Excellence SBSP, University “Politecnica delle Marche” of Ancona.

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Correspondence to Simona Pichini.

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The authors declare that they have no conflicts of interest.

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All procedures performed in the present study were in accordance with the ethical standards of the Università Politecnica delle Marche local committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.

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Published in the topical collection Euroanalysis XX with guest editor Sibel A. Ozkan.

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Mannocchi, G., Di Trana, A., Tini, A. et al. Development and validation of fast UHPLC-MS/MS screening method for 87 NPS and 32 other drugs of abuse in hair and nails: application to real cases. Anal Bioanal Chem (2020).

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  • New psychoactive substances
  • Hair
  • Nails
  • Alternative matrices