Determination of 74 new psychoactive substances in serum using automated in-line solid-phase extraction-liquid chromatography-tandem mass spectrometry

- Rapid qualitative analysis of 95 new psychoactive substances (NPS) in serum.
- Fully validated method for 74 analytes.
- NPS were measured by a fully automated in-line SPE-LC-MS/MS system with a total cycle time of 11 min.
- Required sample volume of 150 mL serum.
- Limit of detection (LOD): 0.2-4 μg/L; lower limit of quantification (LLOQ): 5 μg/L.

A detailed description is given of the development and validation of a fully automated in-line solid-phase extraction-liquid chromatography-tandem mass spectrometry (SPE-LC-MS/MS) method capable of detecting 90 central-stimulating new psychoactive substances (NPS) and 5 conventional amphetamine-type stimulants (amphetamine, 3,4-methylenedioxy-methamphetamine (MDMA), 3,4-methylenedioxy-amphetamine (MDA), 3,4-methylenedioxy-N-ethyl-amphetamine (MDEA), methamphetamine) in serum. The aim was to apply the validated method to forensic samples. The preparation of 150 μL of serum was performed by an Instrument Top Sample Preparation (ITSP)-SPE with mixed mode cation exchanger cartridges. The extracts were directly injected into an LC-MS/MS system, using a biphenyl column and gradient elution with 2 mM ammonium formate/0.1% formic acid and acetonitrile/0.1% formic acid as mobile phases. The chromatographic run time amounts to 9.3 min (including re-equilibration). The total cycle time is 11 min, due to the interlacing between sample preparation and analysis. The method was fully validated using 69 NPS and five conventional amphetamine-type stimulants, according to the guidelines of the Society of Toxicological and Forensic Chemistry (GTFCh). The guidelines were fully achieved for 62 analytes (with a limit of detection (LOD) between 0.2 and 4 μg/L), whilst full validation was not feasible for the remaining 12 analytes. For the fully validated analytes, the method achieved linearity in the 5 μg/L (lower limit of quantification, LLOQ) to 250 μg/L range (coefficients of determination > 0.99). Recoveries for 69 of these compounds were greater than 50%, with relative standard deviations ≤ 15%. The validated method was then tested for its capability in detecting a further 21 NPS, thus totalling 95 tested substances. An LOD between 0.4 and 1.6 μg/L was obtained for these 21 additional qualitatively-measured substances. The method was subsequently successfully applied to 28 specimens from routine forensic case work, of which 7 samples were determined to be positive for NPS consumption.