Optimization of NPS sample preparation in blood, urine, serum, and plasma

New/Novel Psychoactive Substances (NPSs) are newly emerging, rapidly developing compounds designed to mimic the intoxicating effects of illicit substances. The ever-changing landscape of the NPS market and the varying physiochemical properties of these compounds make developing and validating up-to-date analytical methods a challenge. Different extraction and derivatization procedures were assessed to determine an optimal procedure for synthetic cathinones and phenethylamines that could be implemented by forensic toxicology laboratories. Nineteen NPS stimulants and psychedelics were assessed for comparison including NBOMe compounds and synthetic cathinones.

Analytes were prepared neat at 1,000 ng/mL, derivatized using PFPA, MSTFA, TMSI, or TFAA, and incubated at 24, 37, 50, or 70^oC for 20 or 40 minutes. Each combination was assessed in triplicate for a total of 96 samples. After incubation, samples were dried under nitrogen, reconstituted with ethyl acetate, and analyzed by GC-MS. The optimal derivatization procedure was determined by comparing peak areas and fragmentation ion quality. The best procedure overall derivatized with PFPA and incubated at 37^oC for 20 minutes.

An optimal extraction procedure was determined by comparing the following solid phase extraction (SPE) and supported liquid extraction (SLE) cartridges:

• Clean Screen® DAU (200 mg, 6 mL)
• Clean Screen® DAU (130 mg, 3 mL)
• XtrackT® DAU (200 mg, 6 mL)
• XtrackT® Propylsulfonic Acid (500 mg, 10 mL)
• Clean Screen XCEL® I (130 mg, 3 mL)
• Oasis® MCX (150 mg, 6 mL)
• SLE ISOLUTE ® (1 mL)

Triplicates of samples were prepared at 1,000 ng/mL and extracted using each of the six extraction columns. Internal standard was added after elution before samples were dried under nitrogen, derivatized, evaporated again, and reconstituted in ethyl acetate for analysis by GC-MS. The difference in peak area ratio (PAR) for each sample compared to unextracted standards was used to determine extraction efficiency.

All columns tested successfully extracted the 19 analytes except for the XtrackT® Propylsulfonic Acid column which does not have the proper phase chemistry for retention of synthetic cathinones under the conditions used. The Clean Screen® DAU (130 mg, 3 mL) column was the most effective at recovering analytes from blood, plasma, and serum . The Clean Screen XCEL® I (130 mg, 3 mL) column was the most effective at recovering analytes from urine. Extraction efficiencies ranged from 49-119% using the best-performing SPE cartridges.

To further explore extractions for synthetic cathinones and phenethylamines, refer to UCT’s application notes “Analysis of Synthetic Cathinones From Blood and Urine Using Clean Screen® XCEL I on LC-MS/MS” and “Identification and Quantification of Psychedelic Phenethylamine “2C” Series using SPE and LC-MS/MS.”

Citation: Nisbet, L., Wylie, F. M., & Scott, K. S. (2024). Applications of Sample Preparation Techniques in the Analysis of New Psychoactive Substances. Separations, 11(9), Article 258. https://doi.org/10.3390/separations11090258