Determination of new psychoactive substances and other drugs in postmortem blood and urine by UHPLC–MS/MS: method validation and analysis of forensic samples

Abstract

Purpose This study aimed to validate a modified QuEChERS method followed by ultra-high performance liquid chromatography-tandem mass spectrometry to determine 79 new psychoactive substances (NPS) and other drugs in blood and urine. Methods Prescription drugs (n = 23), synthetic cathinones (n = 13), phenethylamines (n = 11); synthetic cannabinoids (n = 8), amphetamines (n = 7) and other psychoactive substances (n = 17) were included in the method. 500 µL of biological fluid was extracted with 2 mL of water/ACN (1:1), 500 mg of anhydrous MgSO 4 /NaOAc (4:1) added, followed by a supernatant cleanup with 25 mg of primary secondary amine and 75 mg of anhydrous MgSO 4 . Quantification was done using matrixmatched calibration curves and deuterated internal standards. ResultsThe method was satisfactorily validated for blood and urine at limit of quantifications ranging from 0.4 to 16 ng/ mL, and applied to the analysis of 54 blood (38 postmortem and 16 antemortem) and 16 antemortem urine samples from 68 forensic cases. All urine samples and 59.3% of the blood samples were positive for at least one analyte. Twenty-two analytes were detected in at least one biological sample, including the synthetic cathinones ethylone (222 ng/mL, antemortem blood), eutylone (246 and 446 ng/mL, urine), and N-ethylpentylone (597 and 7.3 ng/mL, postmortem and antemortem blood, respectively). Conclusions The validated method was shown to be suitable for the analysis of blood and urine forensic samples and an important tool to collect information on emerging drug threats and understanding the impact of NPS and other drugs in poisoning cases.

Fatal cases involving new psychoactive substances and trends in analytical techniques

“…The breakdown of 25R-NBOH family compounds in GC-MS analysis was overcome with shortened columns (4 m-length; Ferrari Júnior et al, 2020 ). GC-MS was not suitable to screen for synthetic cannabinoids ( Hvozdovich et al, 2020 ), fentanyl analogs ( Poklis et al, 2016 ), phenethylamines ( Kristofic et al, 2016 ), and others NPS due to low concentrations detected in intoxications involving these compounds ( Ferrari Júnior and Caldas, 2021 ). Cathinones can exhibit a “poor fragmentation” in the GC-MS, and more specific mass spectra can be obtained using LC-MS or LC-MS/MS ( Mochizuki et al, 2021 ).…”

“…In total, 16 studies included in this review used protein precipitation (PP) as an intermediate or only extraction step, a simple and fast protocol that presents a poor cleanup. Fifty one studies applied liquid-liquid extraction (LLE) using different solvent systems (mostly using alkaline extraction) ( Rojkiewicz et al, 2016 ; Kriikku et al, 2020 ), 36 used solid phase extraction (SPE) columns, after solvent/buffer addition, enzymatic hydrolysis and/or PP ( Rohrig et al, 2017 ; Garneau et al, 2020 ) and five studies used QuEChERS (quick, easy, cheap, effective, rugged, and safe) methods ( Table 1 ), which is a combination of LLE and salts and dispersive SPE with primary and secondary amine (PSA) ( Fujita et al, 2016 ; Ferrari Júnior and Caldas, 2021 ).…”

A review of synthetic cathinones emerging in recent years (2019–2022)

“…in fatal intoxication cases [ng/mL or g] Conc. in non-fatal intoxication cases [ng/mL] Reported intoxication symptoms N- Ethylhexedrone 2-(Ethylamino)-1-phenylhexan-1-one 50–60 Blood: 4–285 Urine: 147–1477 Bile: 34 Lung Brain: 5 [ 97 , 127 , 129 – 133 ] Blood: 1–84 Urine: 165 [ 100 , 127 , 134 – 136 ] Euphoria, stimulation, hyperthermia, tachycardia, vasoconstriction, aggression, dilatated pupils, anxiety, hallucinations, paranoia, acute kidney failure, insomnia, convulsions Ephylone 1-(1,3-Benzodioxol-5-yl)-2-(ethylamino)-pentan-1-one 10–100 Blood: 1–50,000 Urine: 18,000 [ 9 , 58 – 62 , 133 , 143 – 146 ] Blood: 7–149 Urine: 2000–10,700 Oral fluid: 13–1380 [ 58 , 143 , 147 , 148 ] Euphoria, stimulation, tachycardia, hyperthermia, palpitations, delusional state, aggression, psychosis, paranoia, hallucinations, confusion, sleeplessness, cardiac arrest, dissociative effects, acidosis, mydriasis rhabdomyolysis, renal failure Eutylone 1-(1,3-Benzodioxol-5-yl)-2-(ethylamino)-butan-1-one 50–200 Blood: 1–11,000 Urine: 192,000 Gastric content: 2120 Fat tissue: 1310 [ 65 , 67 , 150 ] Blood: 1–3600 Urine: 54–18,400 [ 65 , 67 , 151 – 154 ] ...…”

“…Based on its pharmacology, eutylone has a greater potential for abuse and dose-dependent locomotor stimulation in rodents as compared with its structural isomers pentylone and dibutylone [67,149]. As a result of the popularity of this compound, there are many reports of fatal [65,67,150] and non-fatal [65,67,[151][152][153][154] poisonings, which were confirmed by detecting eutylone in blood. The concentration ranges were 1-11,000 ng/mL in fatal cases and 1-3600 ng/ mL in non-fatal cases (Table 1).…”

Psychoactive substances use before suicide: detailed analysis of all cases that occurred in the Brazilian Federal District in a 10-years period

“…These effects, alone or combined with the simultaneous use of PASs, may mediate suicide-related behaviors; alcohol/drug addiction is also associated with a higher risk of dying by suicide [11]. The main analytical techniques used to perform the toxicological tests to detect the use of these PASs among those who die are Gas Chromatography-Mass Spectrometry (GC-MS) and High-Performance Liquid Chromatography (HPLC) [12,13].…”