Development and Validation of a GC-MS-EI Method to Determine α-PHP in Blood: Application to Samples Collected During Medico-Legal Autopsies.

New Psychoactive Substances (NPS) still represent an issue of great concern worldwide despite efforts made by national and international control systems to limit the spread of these substances. Alpha-pyrrolidinohexanophenone (α-PHP) is a fairly recent synthetic cathinone (the second largest group of monitored substances in Europe) with only a few published studies on the substance. Though there is a low incidence of NPS consumption in Portugal, a recent increase in apprehensions and detection in biological matrices of the substance was verified. An analytical methodology was developed and validated for determining and quantitating α-PHP in blood. Solid phase extraction was employed for sample preparation (500 μL), further analysed by GC-MS-EI in single ion monitoring mode, with cocaine-d3 as the internal standard. Method validation followed the guidelines of the American National Standards Institute/AAFS Standards Board (ANSI/ASB Standard 036). The procedure was linear between 10 and 1000 ng/mL, with determination coefficients (r2) higher than 0.999. Carryover was not observed. A limit of detection of 5ng/mL and a limit of quantitation of 10 ng/mL were achieved. Intraday and intermediate precision and bias assessment showed satisfactory results (CV <17.7%; bias <11.6%), and extraction efficiency ranged from 98.5% to 103.3%. The stability of the substance was considered acceptable for at least 6h at room temperature, 48h in the autosampler, and 21 days after five freeze/thaw cycles. The developed methodology was applied to fifteen real samples from the Laboratory of Chemistry and Forensic Toxicology, Centre Branch, of the National Institute of Legal Medicine and Forensic Sciences, Portugal, with drug concentrations ranging from 15 ng/mL to 227 ng/mL. Available information for each case is also detailed in the present article.