Multicomponent LC–MS/MS screening method for detection of new psychoactive drugs, legal highs, in urine—Experience from the Swedish population

The advent of new not yet legally regulated psychoactive substances sold over the Internet has created a challenge for clinical toxicology and drug testing laboratories. The routine use of immunoassay screening may no longer be the optimal solution in many instances since the number of analytes covered is becoming insufficient. The aim of this work was to design, validate and apply a multi-component LC–MS/MS method suitable for screening of a large number of target analytes belonging to the class of new psychoactive substances – legal highs. The analytical method was using a five-fold dilution of urine with internal standard (pethidine-d5) and injection of 2 μL. The chromatographic system was using a 1.7-μm 100 mm × 2.1 mm Ethylene Bridged Hybrid (BEH) C18 column and gradient elution with a flow rate of 600 μL/min. Solvent A consisted of 0.1% formic acid and Solvent B was 100% acetonitrile. The gradient elution application was designed to have a wide polarity coverage with total run time of 4.0 min. The tandem mass spectrometer was using an electrospray interface and operated in positive mode. Selected reaction monitoring of two ion transitions was used for each of 26 analytes. Method validation demonstrated limited influence from urine matrix, linear response within the measuring range (0.1–10 μg/mL), acceptable imprecision in quantification (CV < 15%). Some analytes were found not to be stable in urine upon storage. The method was successfully applied in routine drug testing. A total of 87 positive samples with 100 analytical findings were found to contain O-desmethyl-cis-tramadol (mostly without mitragynine), methylenedioxypyrovalerone, 4-fluoroamphetamine, methoxetamine, desoxypipradol, 4-fluoromethcathinone, 5,6-methylenedioxy-2-aminoindane, 4-methylmethcathinone, 3-fluoromethcathinone, 4-hydroxy-N-methyl-N-ethyltryptamine, α-methylamino-butyrophenone and 4-methoxymethcathinone.