Development and validation of a UHPLC-HRMS-QTOF method for the detection of 132 New Psychoactive Substances and synthetic opioids, including fentanyl, in Dried Blood Spots

Dried Blood Spots (DBS) represents a promising micro-sampling technique in the field of forensic toxicology to carry out minimally invasive blood sample collection. In DBS, cheap, fast and easy sampling is combined with effortless store and transport. These properties aimed us to develop and validate a quick and easy procedure for the detection of a large and diverse range of emerging and alarming New Psychoactive Substances (NPS). A drop of whole blood sample was collected on a DBS card and dried for 3 h, from which a total of 132 analytes (including NPS, synthetic opioids NSO and metabolites) plus 13 deuterated internal standards could be extracted using 500 μL of a methanol/acetonitrile mixture (3:1, v/v) and subsequently separated and identified by means of ultra-high-performance liquid-chromatography (UHPLC) coupled to high resolution mass spectrometry (HRMS). The extraction efficiency proved to be reproducible with yields ranging from 30% to 100% depending on the different classes of drugs. Trueness, repeatability, and intermediate precision fulfilled acceptance criteria for almost all synthetic opioids, cathinones and hallucinogens (bias and CV% below ±20%); in particular, the aggregate inter-day trueness data showed extremely limited deviation from the expected concentrations (-10% < bias% < +10%) for 114 target analytes out of 132. The calculated limits of detection ranged from 1.3 to 6.3 ng/mL, consistently exceeding the values experimentally tested. Moderate ion suppression was observed for most analytes, partly caused by blood fortification itself. Good stability of the target analytes at -20 °C, 4 °C, and 35 °C on DBS cards after drying was observed, even for long periods of time. Optimal storage condition appeared to be at 4 °C resulting in virtually no drugs degradation for up to 40 days. The novel analytical method based on DBS sampling, verified on venous whole blood real samples previously tested positive with our routine procedure, conveys remarkable potential in analytical toxicology, clinical analysis, and doping control.